PNA monomer and precursor

ABSTRACT

This application relates to monomers of the general formula (I) for the preparation of PNA (peptide nucleic acid) oligomers and provides method for the synthesis of both predefined sequence PNA oligomers and random sequence PNA oligomers:  
                 
 
     wherein  
     R1, R2, R3, R4, R5 is independently H, halogen, C 1 -C 4  alkyl, nitro, nitrile, C 1 -C 4  alkoxy, halogenated C 1 -C 4  alkyl, or halogenated C 1 -C 4  alkoxy, wherein at least one of R1, R3, and R5 is nitro;  
     R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and  
     B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to monomers suitable for thepreparation of PNA oligomers. The present invention also relates toprecursors to the monomers and methods of making the PNA monomers fromthe precursors. Further, the invention relates to methods of making PNAoligomers using the PNA monomers.

[0003] 2. General Background and State of the Art

[0004] In the last two decades, attempts to optimize the properties ofoligonucleotide by modification of the phosphate group, the ribose ring,or the nucleobase have resulted in a lot of discoveries of newoligonucleotide derivatives for the application in the fields of DNAdiagnostics, therapeutics in the form of antisense and antigene, and thebasic research of molecular biology and biotechnology (U. Englisch andD. H. Gauss, Angew. Chem. Int. Ed. Engl. 1991, 30, 613-629; A. D.Mesmaeker et al. Curt. Opinion Struct. Biol. 1995, 5, 343-355; P. E.Nielsen, Curr. Opin. Biotech., 2001, 12, 16-20.). The most remarkablediscovery is peptide nucleic acid which was reported by the Danish groupof Nielsen, Egholm, Buchardt, and Berg (P. E. Nielsen et al., Science,1991, 254, 1497-1500). PNA is DNA analogue in which anN-(2-aminoethyl)glycine polyamide replaces the phosphate-ribose ringbackbone, and methylene-carbonyl linker connects natural as well asunnatural nucleo-bases to central amine of N-(2-aminoethyl)glycine.Despite radical change to the natural structure, PNA is capable ofsequence specific binding to DNA as well as RNA obeying the Watson-Crickbase pairing rule. PNAs bind with higher affinity to complementarynucleic acids than their natural counterparts, partly due to the lack ofnegative charge on backbone, a consequently reduced charge-chargerepulsion, and favorable geometrical factors (S. K. Kim et al., J. Am.Chem. Soc., 1993, 115, 6477-6481; B. Hyrup et al., J. Am. Chem. Soc.,1994, 116, 7964-7970; M. Egholm et al., Nature, 1993, 365, 566-568; K.L. Dueholm et al., New J. Chem., 1997, 21, 19-31; P. Wittung et al., J.Am. Chem. Soc., 1996, 118, 7049-7054; M. Leijon et al., Biochemistry,1994, 9820-9825.). Also it was demonstrated that the thermal stabilityof the resulting PNA/DNA duplex is independent of the salt concentrationin the hybridization solution (H. Orum et al., BioTechniques, 1995, 19,472-480; S. Tomac et al., J. Am. Chem. Soc., 1996, 118, 5544-5552.). AndPNAs can bind in either parallel or antiparallel fashion, withantiparallel mode being preferred (E. Uhlman et al., Angew. Chem. Int.Ed. Engl., 1996, 35, 2632-2635.).

[0005] A mismatch in a PNA/DNA duplex is much more destabilizing than amismatch in a DNA/DNA duplex. A single base mismatch results in 15° C.and 11° C. lowering of the Tm of PNA/DNA and DNA/DNA, respectively.Homopyrimidine PNA oligomers and PNA oligomers with a highpyrimidine/purine ratio can bind to complementary DNA forming unusuallystable PNA2/DNA triple helices (P. E. Nielsen et al., Science, 1991,254, 1497-1500; L. Betts et al., Science, 1995, 270, 1838-1841; H.Knudsen et al., Nucleic Acids Res., 1996, 24, 494-500.). Although PNAshave amide bond and nucleobases, PNAs show great resistance to bothnuclease and protease. In contrast to DNA, which depurinates ontreatment with strong acids and hydrolyses in alkali hydroxides, PNAsare completely acid stable and sufficiently stable to weak bases.

[0006] Generally, PNA oligomers are synthesized using the wellestablished solid phase peptide synthesis protocol. New strategies formonomers have been developed independently by several groups to optimizePNA oligomer synthesis. The preparation of PNA monomers can be dividedinto the synthesis of a suitably protected N-aminoethylglycine and asuitably protected nucleobase acetic acid derivatives, which is followedby coupling both.

[0007] The first synthetic strategy reported for PNA oligomer synthesiswas Merrifield solid phase synthesis using t-Boc/benzyloxycarbonylprotecting group strategy wherein the backbone amino group protectedwith the t-Boc and the exocyclic amino groups of the nucleobases areprotected with the benzyloxycarbonyl (P. E. Nielsen et al., Science,1991, 254, 1497-1500; M. Egholm et al., J. Am. Chem. Soc., 1992, 114,9677-9678; M. Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895-1897; M.Egholm et al., J. Chem. Soc. Chem. Commun., 1993, 800-801; K. L. Dueholmet al., J. Org. Chess., 1994, 59, 5767-5773; WO 92/20702). PNA monomersprotected with t-Boc/benzyloxycarbonyl are now commercially availablebut are inconvenient to use because repeated treatment with TFA isrequired for t-Boc deprotection and the harsh HF ortrifluoromethanesulfonic acid treatment required for cleavage from theresin and deprotection of benzyloxycarbonyl group from exocyclic amineof nucleobases. Thus this strategy is not compatible with the synthesisof many types of modified PNA oligomers such as PNA-DNA chimera.Furthermore, the use of hazardous acids such as HF ortrifluoromethanesulfonic acid is not commercially embraced in view ofsafety concerns for the operator and the corrosive effect on automationequipment and lines. In addition, the t-Boc/benzyloxycarbonyl protectionstrategy is differential strategy which is defined as a system ofprotecting groups wherein the protecting groups are removed by the sametype of reagent or condition, but rely on the different relative ratesof reaction to remove one group over the other. For example, in thet-Boc/benzyloxycarbonyl protecting strategy, both protecting groups areacid labile, but benzyloxycarbonyl group requires a stronger acid forefficient removal. When acid is used to completely remove the more acidlabile t-Boc group, there is a potential that a percentage ofbenzyloxycarbonyl group will also be removed contemporaneously.Unfortunately, the t-Boc group must be removed from amino group ofbackbone during each synthetic cycle for the synthesis of oligomer. ThusTFA is strong enough to prematurely deprotect a percentage of the sidechain benzyloxycarbonyl group, thereby introducing the possibility ofoligomer branching and reducing the overall yield of desired product.

[0008] In another effort to find a milder deprotecting method for PNAoligomer synthesis that would be compatible with DNA oligomer synthesis,several research groups have developed PNA monomers protected withMmt/acyl wherein the backbone amino group protected with the Mmt and theexocyclic amino groups of the nucleobases are protected with an acylgroup such as benzoyl, anisoyl, and t-butyl benzoyl for cytosine andadenine, or isobutyryl, acetyl for guanine (D. W. Will et al.,Tetrahedron, 1995, 51, 12069-12082; P. J. Finn et al., Nucleic AcidResearch, 1996, 24, 3357-3363; D. A. Stetsenko et al., Tetrahedron Lett.1996, 3571-3574; G.. Breipohl et al., Tetrahedron, 1997, 14671-14686.).

[0009] Alternative PNA monomers protected withFmoc/benzhydryloxycarbonyl are also commercially available wherein thebackbone amino group protected with the Fmoc and the exocyclic aminogroups of the nucleobases are protected with the benzhydryloxycarbonyl(J. M. Coull, et al., U.S. Pat. No. 6,133,444). ButFmoc/benzhydryloxycarbonyl strategy has several drawbacks such as sidereaction during Fmoc deprotection and instability of monomer insolution. The most important side reaction is the migration of thenucleobase acetyl group from the secondary amino function to the freeN-terminal amino function of aminoethylglycine backbone under Fmocdeprotection condition (L. Christensen et al., J. Pept. Sci. 1995,1,175-183 ). The N-acetyl transfer reactions in every cycles duringoligomer synthesis result in accumulation of side products which arehard to separate due to similar polarity and same molecular weight. Alsothe Fmoc protecting group is very unstable in the presence of traceamine. Thus the selection of the solvent for the PNA monomers should becautious. Generally, N-methylpyrrolidone of high quality is recommended.This requires higher cost in the synthesis of PNA oligomer.

[0010] The synthesis of PNA oligomers using Fmoc/benzyloxycarbonyl (S.A. Thomson et al., Tetrahedron, 1995, 6179-6194.) and Fmoc/Mmt (G..Breipohl et al., Bioorg. Med. Chem. Lett., 1996, 6, 665-670.) protectedmonomer has also been reported. However, all of these methods haveserious drawbacks in terms of monomer solubility and preparation, harshreaction condition, and side reactions either during monomer synthesisand/or PNA oligomer synthesis.

[0011] In other efforts to find new monomers, cyclic monomers werereported by ISIS and Biocept. The first strategy developed by ISISreplaces protected backbone by morpholinone (U. S. Pat. No. 5,539,083 ),but the strategy has serious drawback in that the hydroxy functionalgroup generated by coupling reaction should be converted to aminefunctional group in every elongation step during oligomer synthesis.Alternatively, the protected aminoethylglycine part is replaced byN-t-Boc-piperazinone (WO 00/02899). But this strategy also has severaldrawbacks in terms of monomer reactivity in oligomerization and the sameproblems as seen in linear t-Boc strategy as described above.

[0012] Despite recent advances, there remains a need for new monomerthat increases yield, lowers synthetic cost, and is suitable forautomatic and parallel synthesis.

SUMMARY OF THE INVENTION

[0013] The present invention provides novel monomers for increasedefficiency and convenience during synthesis of PNA oligomers. Anotherobject is to provide PNA monomers that can be conveniently applied toinstrumentation such as automated synthesizer for synthesis of PNAoligomers. The novel monomers according to the present invention arecompounds having general formula I:

[0014] wherein

[0015] R1, R2, R3, R4, R5 may be independently H, halogen such as F, Cl,Br or I, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated (such asF and Cl) C₁-C₄ alkyl, or halogenated (such as F and Cl) C₁-C₄ alkoxy,wherein

[0016] at least one among R1, R3 and R5 is nitro,

[0017] R6 may be H or protected or unprotected side chain of natural orunnatural a-amino acid, and

[0018] B is a natural or unnatural nucleobase, wherein when saidnucleobase has an exocyclic amino function, said function is protectedby protecting group which is labile to acids but stable to weak tomedium bases.

[0019] The present invention further provides for methods of preparingcompounds of general formula I from compounds of general formula V.

[0020] In another embodiment, the invention provides for compounds ofgeneral formula V and their preparation methods from compounds ofgeneral formula II.

[0021] wherein

[0022] R1, R2, R3, R4, R5 may be independently H, halogen such as F, Cl,Br or I, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated (such asF and Cl) C₁-C₄ alkyl, or halogenated (such as F and Cl) C₁-C₄ alkoxy,wherein

[0023] at least one among R1, R3, and R5 is nitro,

[0024] R6 may be H or protected or unprotected side chain of natural orunnatural α-amino acid,

[0025] R7 may be H, (C₁-C₄) alkyl, or aryl, and

[0026] B is a natural or unnatural nucleobase, wherein when saidnucleobase has an exocyclic amino function, said function is protectedby protecting group which is labile to acids but stable to weak tomedium bases.

[0027] The present invention further provides methods of preparingcompounds of formula I from compounds of general formula IV.

[0028] The present invention also provides compounds of general formulaIV and their free acid form, and their preparation methods:

[0029] wherein

[0030] R1, R2, R3, R4, R5, and R6 are as defined above, and

[0031] HY is organic or inorganic acid.

[0032] The present invention further provides methods of preparingcompounds of general formula IV from compounds of general formula II.

[0033] Also, the present invention provides compounds of formula II andtheir preparation methods:

[0034] wherein

[0035] R1, R2, R3, R4, R5, R6, and R7 are as defined above.

[0036] The invention is directed to a compound of formula I, for whichits residues are defined above. In particular, the R6 residue may be Hor protected or unprotected side chain of natural α-amino acid. Inanother embodiment, B may be thymine (T), cytosine (C), adenine (A), orguanine (G). Further in particular, the protecting group of B may bebenzyloxycarbonyl or benzhydryloxycarbonyl. In other embodiments, themonomer compound of formula I may have the following configuration: R1is nitro and R3 is halogen, trifluoromethyl, or methyl; and R2, R4, R5are H. Or, R1 is nitro, R3 is Cl, R5 is Cl or methyl, and R2 and R4 areH. Still further, R3 is nitro, and R1, R2, R4 and R5 are H. Yet further,R3 is nitro, R1 is Cl, and R2, R4 and R5 are H.

[0037] The invention is also directed to a method of making the compoundof formula I, comprising cyclizing a compound of formula VI (FIG. 6) inthe presence of a coupling reagent that is customarily used in peptidesynthesis or mixed anhydride. The residues for formula VI is definedabove.

[0038] The invention is also directed to a method of making the compoundof formula I, comprising coupling reaction of a compound of formula IVwith a nucleobase acetic acid moiety in the presence of non-nucleophilicorganic base and a coupling reagent that is customarily used in peptidesynthesis.

[0039] The invention is directed to a compound of formula V, for whichits residues are defined above. In particular, the R6 residue may be Hor protected or unprotected side chain of natural α-amino acid. Inanother embodiment, B may be thymine (T), cytosine (C), adenine (A), orguanine (G). Further in particular, the protecting group of B may bebenzyloxycarbonyl or benzhydryloxycarbonyl. In other embodiments, thecompound of formula V may have the following configuration: R1 is nitroand R3 is halogen, trifluoromethyl, or methyl; and R2, R4, R5 are H. Or,R1 is nitro, R3 is Cl, R5 is Cl or methyl, and R2 and R4 are H. Stillfurther, R3 is nitro, and R1, R2, R4 and R5 are H. Yet further, R3 isnitro, R1 is Cl, and R2, R4 and R5 are H. And yet further, R7 may bemethyl, ethyl, or t-butyl.

[0040] In another embodiment, the invention is directed to a method ofmaking the compound of formula V, comprising coupling reaction of acompound of formula II with a nucleobase acetic acid moiety in thepresence of non-nucleophilic organic base and a coupling reagent that iscustomarily used in peptide synthesis.

[0041] The invention is directed to a compound of formula II, for whichits residues are defined above. In particular, the R6 residue may be Hor protected or unprotected side chain of natural α-amino acid. In otherembodiments, the compound of formula II may have the followingconfiguration: R1 is nitro and R3 is halogen, trifluoromethyl, ormethyl; and R2, R4, R5 are H. Or, R1 is nitro, R3 is Cl, R5 is Cl ormethyl, and R2 and R4 are H. Still further, R3 is nitro, and R1, R2, R4and R5 are H. Yet further, R3 is nitro, R1 is Cl, and R2, R4 and R5 areH.

[0042] The invention is also directed to a method of making the compoundof formula II, comprising reacting 2-aminoethyl sulfonylamide derivativewith haloacetate derivative by a nucleophilic substitution reaction inthe presence of non-nucleophilic organic base.

[0043] The invention is further directed to a compound having formula IVand its free acid form. The residues for formula IV are defined above.But in particular, the R6 residue may be H or protected or unprotectedside chain of natural a-amino acid. In other embodiments, the compoundof formula IV may have the following configuration: R1 is nitro and R3is halogen, trifluoromethyl, or methyl; and R2, R4, R5 are H. Or, R1 isnitro, R3 is Cl, R5 is Cl or methyl, and R2 and R4 are H. Still further,R3 is nitro, and R1, R2, R4 and R5 are H. Yet further, R3 is nitro, R1is Cl, and R2, R4 and R5 are H. Further, HY may be HCl or TFA.

[0044] The invention is further directed to a method of making thecompound of formula IV, comprising cyclizing a compound of formula IIIin the presence of a coupling reagent that is customarily used inpeptide synthesis or mixed anhydride, followed by deprotection of t-Bocin acid.

[0045] The invention is also directed to a method of making PNAoligomer, comprising linking together the compound of formula I.

[0046] It is to be understood that the “R” group designations citedabove apply to all of the compounds of formulae I-VI, including the Rgroups that are cited as particular embodiments. It is also to beunderstood that the R group designations apply to the compounds as theyundergo the processes of the invention.

[0047] These and other objects of the invention will be more fullyunderstood from the following description of the invention, thereferenced drawings attached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

[0049]FIG. 1 shows a chart of the chemical structures of naturally andnon-naturally occurring nucleobases useful for DNA recognition.

[0050]FIG. 2 shows another chart of the chemical structures of naturallyand non-naturally occurring nucleobases useful for DNA recognition.

[0051]FIG. 3 shows a schematic representation of the synthesis ofprotected backbone.

[0052]FIG. 4 shows a schematic representation of the alternativesynthesis of protected backbone.

[0053]FIG. 5 shows a schematic representation of the synthesis ofprotected piperazinone as a precursor for monomer

[0054]FIG. 6 shows a schematic representation of the synthesis of PNAmonomer.

[0055]FIG. 7 shows a schematic representation of the alternativesynthesis of PNA monomer.

[0056]FIG. 8 shows a schematic representation of the synthesis of PNAthymine monomer.

[0057]FIG. 9 shows a schematic representation of the alternativesynthesis of PNA thymine monomer.

[0058]FIG. 10 shows a schematic representation of the synthesis of PNAcytosine monomer

[0059]FIG. 11 shows a schematic representation of the alternativesynthesis of PNA cytosine monomer.

[0060]FIG. 12 shows a schematic representation of the synthesis of PNAadenine monomer.

[0061]FIG. 13 shows a schematic representation of the alternativesynthesis of PNA adenine monomer.

[0062]FIG. 14 shows a schematic representation of the synthesis of PNAguanine monomer.

[0063]FIG. 15 shows a schematic representation of the alternativesynthesis of PNA guanine monomer.

[0064]FIG. 16 shows a schematic representation of the PNA oligomersynthesis from PNA monomers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] In the present invention, nitrobenzenesulfonyl group of acompound having general formula I plays an important role not only as aprotecting group of amine of backbone but also as activating group forcoupling reaction. The monomers having described characteristics areuseful for the synthesis of PNA oligomers by manual or automatedsynthesizer and the preparation of PNA oligomer library by combinatorialchemistry. Nucleobase B in the general formula I is naturally attachedat the position found in nature, i.e., position I for thymine orcytosine, and position 9 for adenine or guanine, as well as fornon-naturally occurring nucleobase (nucleobase analog), or nucleobasebinding moiety. Some nucleobases and illustrative synthetic nucleobasesare shown in FIG. 1 and FIG. 2.

[0066] Preparation of Protected Backbones

[0067] The first step for the preparation of novel monomers havinggeneral formula I is synthesis of[2-(nitrobenzenesulfonylamino)-ethyl]-glycine derivatives having theformula II:

[0068] The entities represented by R1, R2, R3, R4, R5, R6, and R7 are asdefined above.

[0069] With reference to FIG. 3, preferredN-[2-(nitrobenzenesulfonylamino)-ethyl]-glycine derivative issynthesized by mono-protection reaction of ethylenediamine withnitrobenzenesulfonyl chloride, followed by nucleophilic substitutionreaction of the resultant product with haloacetate in the presence ofnon-nucleophilic organic base. Nitrobenzenesulfonyl chlorides areobtained commercially or prepared by known methods such as described inU.S. Pat. No. 4,204,870. Nitrobenzenesulfonyl chlorides the compound ofthe general formula:

[0070] The entities represented by R1, R2, R3, R4, and R5 are as definedabove.

[0071] Haloacetate is a compound of the general formula:

[0072] wherein

[0073] X is a halogen group, such as Cl, Br and I, and

[0074] R7 is as defined above.

[0075] With reference to FIG. 3, the mono-sulfonylation reaction can beconducted by slow addition of nitrobenzenesulfonyl chloride to asolution of excess ethylenediamine in appropriate solvent withoutorganic base. Examples of solvents of above reaction are toluene,benzene, ethylacetate, tetrahydrofuran, diisopropylether, diethyl ether,dichloromethane, chloroform, carbon tetrachloride, and acetonitrile.Preferred solvent is dichloromethane. After completion of the reaction,the reaction mixture is washed with brine, dried over anhydrous sodiumsulfate, and filtered. The desired product is solidified by addingacetic acid to the filtrate. Nucleophilic substitution reaction iscarried out by adding haloactate to the mixture of the mono-sulfonylatedproduct and non-nucleophilic organic base in appropriate solvent.Examples of solvents of above reaction are ethylacetate,tetrahydrofuran, dichloromethane, chloroform, DMF, andN-methylpyrrolidone. Preferred solvent is dichlomethane. Examples ofnon-nucleophilic organic bases include, but are not limited to,triethylamine, tripropylamine N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is triethylamine.

[0076] Alternatively, preferredN-[2-(nitrobenzenesulfonylamino)-ethyl]-glycine derivative issynthesized by selective reaction of primary amine of 2-aminoglycinederivatives, prepared by known methods (for instance, where R1 is H, seeS. A. Thomson et al., Tetrahedron, 1995, 6179-6194; where R1 is a sidechain of a protected or unprotected natural or unnatural a amino acids,see A. Puschl et al., Tetrahedron Lett., 1998, 39, 4707-4710), withnitrobenzenesulfonyl chloride (FIG. 4). The coupling reaction for thepreparation of N-[2-(nitrobenzenesulfonylamino)-ethyl]-glycinederivatives is performed by slow addition of nitrobenzenesulfonylchloride to a solution of N-(2-aminoethyl)-glycine derivatives in thepresence of non-nucleophilic organic base at ambient temperature.Examples of solvents of above reaction are water, toluene, benzene,ethylacetate, tetrahydrofuran, diisopropylether, diethylether,dichloromethane, chloroform, carbon tetrachloride, and acetonitrile.Preferred solvent is dichloromethane. Examples of non-nucleophilicorganic bases include, but are not limited to, triethylamine,tripropylamine N,N-diisopropylethylamine, N-methylmorpholine, andN-ethylmorpholine. Preferred non-nucleophilic organic base istriethylamine. After completion of the reaction by monitoring by thinlayer chromatography (TLC), the reaction mixture is washed with water,dried, and evaporated in reduced pressure to give the desired product.

[0077] Preparation of 1-(Nitrobenzenesulfonyl)-piperazin-2-ones

[0078] The first precursory synthons having formula IV for synthesis ofmonomers having general formula I are prepared fromN-[2-(nitrobenzenesulfonylamino)-ethyl]-glycine derivatives having theformula II by hydrolysis, protection of secondary amine, cyclization,and deprotection of protecting group of secondary amine (FIG. 5).

[0079] The entities represented by R1, R2, R3, R4, R5, R6, and HY are asdefined above.

[0080] First, N-[2-(nitrobenzenesulfonylamino)-ethyl]-glycinederivatives having the formula II are converted to corresponding acidsby adding excess hydroxide ion source. Preferred R2 is methyl or ethylradical. Examples of hydroxide ion sources include, but are not limitedto, lithium hydroxide, sodium hydroxide, and potassium hydroxide.Preferred hydroxide ion source is lithium hydroxide. Then the reactionmixture without work-up is treated with di-t-butyl dicarbonate toprotect secondary amine to afford having the general formula:

[0081] The entities represented by R1, R2, R3, R4, R5, and R6 are asdefined above.

[0082] Preferred hydrolysis reaction is carried out by adding an aqueoussolution of lithium hydroxide (2 equivalent) to a solution ofN-[2-(nitrobenzenesulfonylamino)-ethyl]-glycine derivative at ambienttemperature. After completion of the reaction by TLC analysis, anaqueous solution of lithium hydroxide (additional 1 equivalent) is addedto the reaction mixture. The reaction mixture is stirred for sufficienttime. Then the excess di-t-butyl dicarbonate is removed by extractionwith ethylacetate. Then the aqueous solution is acidified, extractedwith dichloromethane, dried, and evaporated in reduced pressure to yielda solid. Examples of solvents of above reaction are aqueoustetrahydrofuran, aqueous dioxane, and aqueous 1,2-dimethoxyethane.Preferred solvent is aqueous tetrahydrofuran.

[0083] Second, the cyclization reaction of carboxylic acids havinggeneral formula III and followed by deprotection of t-Boc producespiperazinone derivatives having general formula IV The cyclizationreaction occurs simultaneously during activation of carboxylic acid. Theactivation of carboxylic acid can be conducted by general couplingreagent for peptide synthesis at ambient temperature. Examples ofcoupling reagents include, but are not limited to, HATU (L. A. Carpinoet al., J. Am. Chem. Soc., 1993, 115, 4397-4398), HAPyU, TAPipU (A.Ehrlich et al., Tetrahedon Lett., 1993, 4781-4784), HBTU (V. Dourtoglouet al., Synthesis, 1984, 572-574), TBTU, TPTU, TSTU, TNTU (R. Knorr etal., Tetrahedron Lett., 1989, 1927-1930), TOTU, BOP (B. Castro et al.,Tetrahedron Lett., 1975, 1219-1222), PyBOP (J. Coste et al., TetrahedronLett., 1990, 205-208), BroP (J. Coste et al., Tetrahedron Lett., 1990,669-672), PyBroP (J. Coste et al., Tetrahedron Lett., 1991, 1967-1970),BOI (K. Akaji et al., Tetrahedron Lett., 1992, 3177-3180), MSNT (B.Blankemeyer-Menge et al., Tetrahedron Lett., 1990, 1701-1704), TDO (R.Kirstgen et al., J. Chem. Soc. Chem. Commun., 1987, 1870-1871), DCC,EDC. The solvents can be selected from tetrahydrofuran, dichloromethane,chloroform, DMF, and N-methylpyrrolidone. Preferred solvent is DMF.

[0084] Alternatively, the activation of carboxylic acid can be conductedby formation of mixed anhydride using alkyl chloroformate or alkanoylchloride with non-nucleophilic organic base. Examples of alkylhaloformates or alkanoyl chlorides include, but are not limited to,methyl chloroformate, ethyl chloroformate, propyl chloroformate, butylchloroformate, isobutyl chloroformate, pivaloyl chloride, and adamantinecarboxyl chloride. The most preferred acid chloride is isobutylchloroformate. The cyclization reaction using isobutyl chloroformate iscarried out by slowly adding isobutyl chloroformate to a reactionsolution of carboxylic acid having general formula III andnon-nucleophilic organic base in an anhydrous appropriate solvent at thetemperature between −20 ° C. and 0° C. Examples of non-nucleophilicorganic bases include, but are not limited to, triethylaamine,tripropylamine, N,N-diisopropylethylamine, N-methylmorpholine, andN-ethylmorpholine. Preferred non-nucleophilic organic base isN-methylmorpholine. Examples of anhydrous appropriate solvents include,but are not limited to, acetonitrile, chloroform, dichloromethane,1,2-dimethoxy ethane, diethyl ether, diisoproyl ether, andtetrahydrofuran. Preferred solvents are dichloromethane andtetrahydrofuran. The most preferred reaction temperature is that thereaction mixture is allowed to slowly warm to 0° C. after completingaddition of isobutyl chloroformate at −20° C.

[0085] With reference to FIG. 5, the t-Boc group is deprotected in thepresence of acid. Examples of acids include, but are not limited to,HCl, HBr, HF, HI, nitric acid, sulfuric acid, methanesulfonic acid, TFA,and trifluoromethanesulfonic acid. Preferred acid is HCl. The solventsof deprotecting reaction include dichloromethane, chloroform, carbontetrachloride, ethyl acetate, toluene, and benzene. The most preferredsolvent is dichloromethane.

[0086] Synthesis of PNA Monomer

[0087] According to a method of this invention, PNA monomers havinggeneral formula I can be synthesized by at least two methods. Withreference to FIG. 6, the first approach to PNA monomers is a method thatintroduces protected or unprotected nucleobase acetic acid moieties toprotected linear backbone prior to cyclization reaction. Alternatively,PNA monomers can be synthesized by beginning with cyclization ofprotected linear backbone, followed by coupling of protected orunprotected nucleobase acetic acid moieties to create desired products.

[0088] Method 1

[0089] The linear moieties having general formula V are prepared fromprotected linear backbone having general formula II by acylation ofnucleobase acetic acid moieties using coupling reagents as shown in FIG.6.

[0090] With reference to FIG. 6, the coupling reaction was conducted byaddition of coupling reagent to the mixture of protected linear backbonehaving general formula II, nucleobase acetic acid moieties, andnon-nucleophilic organic base in anhydrous appropriate solvent. Examplesof coupling reagents include, but are not limited to, HATU HAPyU, TAPip,HBTU, TBTU, TPTU, TSTU, TNTU, TOTU, BOP, PyBOP, BroP, MSNT, TDO, DCC,EDC. Preferred coupling reagent is PyBOP. Examples of non-nucleophilicorganic bases include, but are not limited to, triethylamine,tripropylamine, N,N-diisopropylethylamine, N-methylmorpholine, andN-ethylmorpholine. Preferred non-nucleophilic organic base isdiisopropylethylamine. Examples of anhydrous appropriate solventsinclude, but are not limited to, chloroform, dichloromethane,1,2-dimethoxyethane, tetrahydrofuran, DMF, and N-methylpyrrolidone.Preferred solvent is DMF.

[0091] Compounds having the general formula V are converted tocorresponding acids such as formula VI by adding an excess of hydroxideion source. Preferred R2 is methyl or ethyl radical. Examples ofhydroxide ion sources include, but are not limited to, lithiumhydroxide, sodium hydroxide, and potassium hydroxide. Preferredhydroxide ion source is lithium hydroxide.

[0092] The entities represented by R1, R2, R3, R4, R5, R6, and B are asdefined above.

[0093] With reference to FIG. 6, the cyclization reaction of carboxylicacids produces PNA monomers general formula I by simultaneous reactionduring activation of carboxylic acid. The activation of carboxylic acidcan be conducted by general coupling reagent for peptide synthesis atambient temperature. Examples of coupling reagents include, but are notlimited to, HATU, HAPyU, TAPip, HBTU, TBTU, TPTU, TSTU, TNTU, TOTU, BOP,PyBOP, BroP, MSNT, TDO, DCC, EDC. Preferred coupling reagent is PyBOP.Examples of non-nucleophilic organic bases include, but are not limitedto, triethylamine, tripropylamine, N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is N,N-diisopropylethylamine. The solvents can be selectedfrom tetrahydrofuran, dichloromethane, chlorofonn, DMF, andN-methylpyrrolidone. Preferred solvent is DMF.

[0094] Alternatively, the activation of carboxylic acid can be conductedby formation of mixed anhydride using alkyl chloroformate or alkanoylchloride with non-nucleophilic organic base. Examples of alkylhaloformates or alkanoyl chlorides include, but are not limited to,methyl chloroformate, ethyl chloroformate, propyl chloroformate, butylchloroformate, isobutyl chloroformate, pivaloyl chloride, and adamantinecarboxyl chloride. The most preferred acid chloride is isobutylchloroformate. The cyclization reaction using isobutyl chloroformate iscarried out by slowly adding isobutyl chloroformate to a reactionsolution of carboxylic acid and non-nucleophilic organic base in ananhydrous appropriate solvent at a temperature between −20 ° C. and 0°C. Examples of non-nucleophilic organic bases include, but are notlimited to, triethylamine, tripropylamine, N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is N-methylmorpholine. Examples of anhydrous appropriatesolvents include, but are not limited to, acetonitrile, chlorofonn,dichloromethane, 1,2-dimethoxyethane, diethyl ether, diisoproyl ether,and tetrahydrofuran. Preferred solvents are dichloromethane andtetrahydrofuran. The most preferred reaction temperature is that thereaction mixture is allowed to slowly warm to 0° C. after completingaddition of isobutyl chloroformate at −20° C.

[0095] Method 2

[0096] As an alternative method, PNA monomers according to thisinvention may be prepared by coupling of protected or unprotectednucleobase acetic acid moieties to cyclic precursor having generalformula IV:

[0097] The entities represented by R1, R2, R3, R4, R5, and R6 are asdefined above.

[0098] With reference to FIG. 7, the coupling reaction of cyclicprecursor with protected or unprotected nucleobase acetic acid moietiesis carried out by using general coupling reagent for peptide synthesisand non-nucleophilic organic bases at ambient temperature. Examples ofcoupling reagents include, but are not limited to, HATU, HAPyU, TAPip,HBTU, TBTU, TPTU, TSTU, TNTU, TOTU, BOP, PyBOP, BroP, MSNT, TDO, DCC,EDC. Preferred coupling reagent is PyBOP. Examples of non-nucleophilicorganic bases include, but are not limited to, triethylamine,tripropylamine, N,N-diisopropylethylamine, N-methylmorpholine, andN-ethylmorpholine. Preferred non-nucleophilic organic base isN,N-diisopropylethylamine. The solvents can be selected fromtetrahydrofuran, dichloromethane, chlorofonr, DMF, andN-methylpyrrolidone. Preferred solvent is DMF.

[0099] Nucleobases and Protecting Group

[0100] Examples of nucleobases of this invention include, but are notlimited to, adenine, cytosine, guanine, thymine, uridine,2,6-diaminopurine, and naturally or non-naturally occurring nucleobasesas depicted in FIG. 1 and FIG. 2. Preferred nucleobases are adenine,cytosine, guanine, and thymine. Nucleobases may be protected byprotecting group for the syntheses of PNA oligomers. Protecting groupsmay be, but are not limited to, Boc, adamantyloxycarbonyl,benzyloxycarbonyl (P. E. Nielsen et al., Science, 1991, 254, 1497-1500;M. Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677-9679; M. Egholm etal., J. Am. Chem. Soc., 1992, 114, 1895-1897; M. Egholm et al., J. Chem.Soc. Chem. Commun., 1993, 800-801; K. L. Dueholm et al., J. Org. Chem.,1994, 59, 5767-5773; WO 92/20702), p-methoxybenzyloxycarbonyl,benzhydryloxycarbonyl (U.S. Pat. No. 6,133,444),2-methlylthioethoxycarbonyl (U.S. Pat. No. 6,063,569), Mmt (G. Breipohlet al., Bioorg. Med. Chem. Lett., 1996, 6, 665-670), or acid labileprotecting group (T. W. Greene M. Wuts, Protective Group in OrganicSynthesis, 3^(rd) Edition, pp 494˜653).

[0101] Synthesis of T-Monomer

[0102] T-monomer is a compound having general formula I-t:

[0103] The entities represented by R1, R2, R3, R4, R5, and R6 are asdefined above.

[0104] Preferred monomers are:

[0105] R1 is nitro and R3 may be an electron withdrawing group such asF, Cl, or trifluoromethyl.

[0106] R3 is nitro and R1 or R5 may be an electron withdrawing groupsuch as F or Cl.

[0107] The precursor for T-monomer, (thymin-1-yl)-acetic acid (shownbelow), is prepared by known method (K. L. Dueholm et. al., J. Org.Chem., 1994, 59, 5767-5773; WO 92/20702).

[0108] With reference to FIG. 8, The compounds of general formula V-tare prepared by coupling reaction of (thymin-1-yl)-acetic acid withnitrobenzenesulfonyl group protected backbone ester having generalformula II in the presence of coupling reagent to afford the compoundhaving general formula:

[0109] The entities represented by R1, R2, R3, R4, R5, R6, and R7 are asdefined above.

[0110] Preferred R7 is methyl or ethyl radical.

[0111] The compounds having the general formula V-t are converted tocorresponding acids by adding an excess hydroxide ion source to affordthe compound having general formula VI-t.

[0112] The entities represented by R1, R2, R3, R4, R5, and R6 are asdefined above.

[0113] With reference to FIG. 8, the cyclization reaction of carboxylicacids produces PNA T-monomers having general formula I-t by simultaneousreaction during activation of carboxylic acid. The activation ofcarboxylic acid can be conducted by general coupling reagent for peptidesynthesis or mixed anhydride. The reaction conditions and reagents arethe same as described above.

[0114] Alternatively, as seen in FIG. 9, PNA T-monomers can be preparedby coupling (thymin-1-yl)-acetic acid to piperazinone derivatives havinggeneral formula IV. The reaction conditions and reagents are the same asdescribed above.

[0115] Synthesis of C-Monomer

[0116] C-monomer is a compound having general formula I-c:

[0117] R8 may be benzyl or benzhydryl group.

[0118] The entities represented by R1, R2, R3, R4, R5, and R6 are asdefined above.

[0119] Preferred monomers are:

[0120] R1 is nitro and R3 may be an electron withdrawing group such asF, Cl, or trifluoromethyl. Or,

[0121] R3 is nitro and R1 or R5 may be an electron withdrawing groupsuch as F or Cl.

[0122] The precursors for PNA C-monomers, suitably protected(cytosin-1-yl)-acetic acids (shown below), are prepared by known methodssuch as described in U.S. Pat. No. 6,133,444; U.S. Pat. No. 6,063,569;Dueholm, et al., J. Org. Chem., 1994, 59, 5767-5773; WO 92/20702, whichare incorporated by reference herein in their entirety, or modificationsthereof.

[0123] R8 may be benzyl or benzhydryl group.

[0124] With reference to FIG. 10, PNA C-monomer is prepared by couplingreaction of suitably protected (cytosin-1-yl)-acetic acids with anitrobenzenesulfonyl group protected backbone ester having generalformula II in the presence of coupling reagent to afford the compoundhaving general formula:

[0125] The entities represented by R1, R2, R3, R4, R5, R6, R7, and R8are as defined above.

[0126] The compounds having the general formula V-c are converted tocorresponding acids by adding an excess of hydroxide ion source toafford the compound having general formula VI-c:

[0127] The entities represented by R1, R2, R3, R4, R5, R6, and R8 are asdefined above.

[0128] With reference to FIG. 10, the cyclization reaction of carboxylicacids produces PNA monomers general formula I-c by simultaneous reactionduring activation of carboxylic acid. The activation of carboxylic acidcan be conducted by general coupling reagent for peptide synthesis ormixed anhydride. The reaction conditions and reagents are the same asdescribed above.

[0129] Alternatively, as seen in FIG. 1, PNA C-monomer can be preparedby coupling suitably protected (cytosin-1-yl)-acetic acids topiperazinone derivatives having general formula IV. The reactionconditions and reagents are the same as described above.

[0130] Synthesis of A-Monomer

[0131] A-monomer is a compound having general formula I-a:

[0132] The entities represented by R1, R2, R3, R4, R5, R6, and R8 are asdefined above.

[0133] Preferred monomers are:

[0134] R1 is nitro and R3 may be an electron withdrawing group such asF, Cl, or trifluoromethyl. Or,

[0135] R3 is nitro and R1 or R5 may be an electron withdrawing groupsuch as F or Cl.

[0136] The precursors for PNA A-monomers, suitably protected(adenin-9-yl)-acetic acids (shown below), are prepared by known methodssuch as described in U.S. Pat. No. 6,133,444; and S. A. Thomson et al.,Tetrahedron, 1995, 6179-6194, which are incorporated by reference hereinin their entirety, or modifications thereof.

[0137] R8 is selected from benzyl or benzhydryl group.

[0138] With reference to FIG. 12, PNA C-monomer is prepared by couplingreaction of suitably protected (adenin-9-yl)-acetic acids with anitrobenzenesulfonyl group protected backbone ester having generalformula II in the presence of coupling reagent to afford the compoundhaving general formula V-a:

[0139] The entities represented by R1, R2, R3, R4, R5, R6, R7, and R8are as defined above.

[0140] The compounds having the general formula V-a are converted tocorresponding acids by adding an excess hydroxide ion source to affordthe compound having general formula VI-a:

[0141] The entities represented by R1, R2, R3, R4, R5, R6, and R8 are asdefined above

[0142] With reference to FIG. 12, the cyclization reaction of carboxylicacids produces PNA monomers general formula I-a by simultaneous reactionduring activation of carboxylic acid. The activation of carboxylic acidcan be conducted by general coupling reagent for peptide synthesis ormixed anhydride. The reaction conditions and reagents are the same asdescribed above.

[0143] Alternatively, as seen in FIG. 13, PNA A-monomer can be preparedby coupling suitably protected (adenin-9-yl)-acetic acids topiperazinone derivatives having general formula IV. The reactionconditions and reagents are the same as described above.

[0144] Synthesis of G-Monomer

[0145] G-monomer is a compound having general formula I-g:

[0146] The entities represented by R1, R2, R3, R4, R5, R6, and R8 are asdefined above.

[0147] Preferred monomers are:

[0148] R1 is nitro and R3 may be an electron withdrawing group such asF, Cl, or trifluoromethyl. Or,

[0149] R3 is nitro and R1 or R5 may be an electron withdrawing groupsuch as F or Cl.

[0150] The precursors for PNA G-monomers, suitably protected(guanin-9-yl)-acetic acids (shown below), are prepared by known methodssuch as described in U.S. Pat. No.6,172,226, or modifications thereof.

[0151] R8 may be benzyl or benzhydryl group.

[0152] With reference to FIG. 14, PNA G-monomer is prepared by couplingreaction of suitably protected (guanin-9-yl)-acetic acids with anitrobenzenesulfonyl group protected backbone ester having generalformula II in the presence of coupling reagent to afford the compoundhaving general formula V-g:

[0153] The entities represented by R1, R2, R3, R4, R5, R6, R7, and R8are as defined above

[0154] The compounds having the general formula V-g are converted tocorresponding acids by adding an excess hydroxide ion source to affordthe compound having general formula VI-g:

[0155] The entities represented by R1, R2, R3, R4, R5, R6, and R8 are asdefined above

[0156] With reference to FIG. 14, the cyclization reaction of carboxylicacids produces PNA monomers general formula I-g by simultaneous reactionduring activation of carboxylic acid. The activation of carboxylic acidcan be conducted by general coupling reagent for peptide synthesis ormixed hydride. The reaction conditions and reagents are the same asdescribed above.

[0157] Alternatively, as seen in FIG. 15, PNA G-monomer can be preparedby coupling suitably protected (guanin-9-yl)-acetic acids topiperazinone derivatives having general formula IV. The reactionconditions and reagents are the same as described above.

[0158] Synthesis of PNA Oligomers

[0159] Various combinatorial synthetic methods already reported inchemical literature are generally applicable to PNA oligomer synthesisusing the monomers of this invention. These methods include, but are notlimited to, solid phase synthesis and solution phase synthesis. Afterthe PNA monomers have been synthesized in the manner described above,PNA oligomers are constructed by solid phase synthesis on a suitablesupport material (example, but not limited to, polystyrene,polyoxyethylene-modified polystyrene, such as , for example Tentagel®,Controlled Pore Glass), which is provided with anchoring group whichlatently contains cleavable amine functional group. In solid phasesynthesis, the first PNA monomer of this invention is incorporated bycoupling reaction to solid support. Then the next sep is systematicelaboration of desired PNA oligomer sequence. This elaboration includesrepeated deprotection/coupling/capping cycles. The backbone protectinggroup on the last coupled monomer, nitrobenzenesulfonyl group, isquantitatively removed by treatment with suitable thiol in the presenceof organic base to liberate terminal free amine. Once the synthesis ofPNA oligomer is completed, oligomers are cleaved from the solid supportand nucleobase protecting groups are simultaneously removed byincubation for 1-2h at room temperature in TFA containing cresol as acation scavenger.

[0160] Following is the general cycle used for the synthesis of PNAoligomers:

[0161] 1. Removing protecting group from resin to activate aminefunctional group.

[0162] 2. Incorporating amino-acid, linker, or PNA monomer havingterminal protected amine group to resin.

[0163] 3. Washing.

[0164] 4. Capping with acetic anhydride in the presence of organic base.

[0165] 5. Washing.

[0166] 6. Cleavage over reacted acetyl group in sulfonamide.

[0167] 7. Washing.

[0168] 8. Deprotecting sulfonyl group.

[0169] 9. Washing.

[0170] 10. Adding monomer.

[0171] 11. Returning to No. 3 and repeat No. 4 - No. 11.

[0172] In the course of coupling reaction of monomer for the oligomersynthesis, the acylating reaction can be accelerated by using catalystsuch as mercury acetate, tetramethylammonium fluoride,tetraethylammonium fluoride, tetrabutylammonium fluoride,benzyltrimethylammonium fluoride, cesium fluoride, tributylphosphine,triphenylphosphine. Preferred catalyst is tetrabutylammonium fluoride.Also the reaction rate depends on solvent and temperature. Examples ofsolvents include, but are not limited to, DMF, N-methylpyrrolidone,dimethoxyethane, dichloromethane, 1,2-dichloroethane, DMSO,tetrahydrofuran, hexamethylphophoramide, tetramethylene sulfone,isopropyl alcohol, ethyl alcohol, and mixture of selected sovents.Preferred solvent is DMF. The N-terminal amino protecting group iscleaved by using thiol with organic base in solvent. Examples of thiolsinclude, but are not limited to, C₂C₂₀ alkanethiol,4-methoxytoluenethiol, 4-methylbenzenethiol, 3,6-dioxa-1,8-octanethiol,4-chlorotoluenethiol, benzylmercaptane, N-acetylcysteine,N-(t-Boc)cysteine methyl ester, methyl 3-mercaptopropionate,4-methoxybenzene thiol. Examples of organic bases include, but are notlimited to, triethylamine, N,N-diisopropyethylamine, piperidine,N-methylmorpholine, and 1,8-diazabicyclo[5,4,0]undec-7-one. Preferredorganic base is N,N-diisopropyethylamine.

[0173] List of Abbreviations.

[0174] t-Boc tert-Butyloxycarbonyl

[0175] BOI 2-(Benzotriazol-1-yl)oxy-1,3-dimethyl-imidazoliniumhexafluorophosphate

[0176] BOP Benzotriazolyl-1-oxy-tris(dimethylamino)phophoniumhexafluorophosphate

[0177] BroP Bromotris(dimethylamino)phophonium hexafluorophosphate

[0178] DMF Dimethylformamide

[0179] Fmoc 9-Fluorenylmethyloxycarbonyl

[0180] HAPyUO-(7-Azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uraniumhaxafluorophosphate

[0181] HATU O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluraniumhexafluorophosphate

[0182] HBTU O-(Benzotriazol-1-yl)-1,1,3,3-tetramethyluraniumhexafluorophosphate

[0183] MSNT 2,4,6-Mesitylenesulfonyl-3-nitro-1,2,4-triazolide

[0184] Mmt 4-Methoxyphenyldiphenylmethyl

[0185] PyBOP Benzotriazolyl-1-oxy-tripyrrolidinophosphoniumhexafluorophosphate

[0186] PyBroP Bromotripyrrolidinophosphoniium hexafluorophosphate

[0187] TAPipUO-(7-Azabenzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uraniumtetrafluoroborate

[0188] TBTU O-(Benzotriazol-1-yl)-1,1,3,3 -tetramethyluraniumtetrafluoroborate

[0189] TDO 2,5-Diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide

[0190] TFA Trifluoroacetic acid

[0191] TNTUO-[(5-Norbonene-2,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate

[0192] J TOTUO-[(Cyano(ethoxycarbonyl)methylene)amino]-1,1,3,3-tetramethyluroniumtetrafluoroborate

[0193] TPTU O-(1,2-Dihydro-2-oxo-1-pyridyl-1,1,3,3-tetramethyluroniumtetrafluoroborate

[0194] TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate

[0195] This invention is more specifically illustrated by followingExamples, which are not meant limit the invention, unless otherwisenoted.

EXAMPLES Example 1 N-(2-Aminoethyl)-2-nitro-benzenesulfonamide aceticacid salt

[0196] 1.2-Diaminoethnae (60.1 g, 1.00 mol) was dissolved indichloromethane (1 L). A solution of 2-nitro-benzenesulfonyl chloride(22.16 g, 0.100 mol) in dichloromethane (100 mL) was added dropwise over3h. After additional stirring for 30 min, the reaction mixture waswashed with brine (1L×4). The organic layer was dried over magnesiumsulfate and filtered. To the filtrate was added acetic acid (6.0 g, 0.1mol) to precipitate solid. The solid was filtered off, washed withdichloromethane (200 mL), and dried in vacuo to afford the titlecompound as a yellow solid (23.1 g, 76%). ¹H-NMR (500 MHz; DMSO-d₆) δ8.01˜7.95 (m, 2H), 7.88˜7.83 (m, 2H), 2.90 (t, 2H), 2.60 (t, 2H), 1.87(s, 3H).

Example 2 N-(2-Aminoethyl)-4-chloro-2-nitro-benzenesulfonamide aceticacid salt

[0197] The title compound (22.5 g, 66%) was synthesized from4-chloro-2-nitro-benzenesulfonyl chloride (25.61 g, 0.1 mol) as per theprocedure of example 1. ¹H-NMR (500 MHz; DMSO-d₆) δ 8.26 (d, 1H),8.01˜7.97 (m, 2H), 2.97 (t, 2H), 2.69 (t, 2H), 1.91 (s, 3H).

Example 3 N-(2-Aminoethyl)-4-fluoro-2-nitro-benzenesulfonamide aceticacid salt

[0198] The title compound (21.7 g, 67%) was synthesized from4-fluoro-2-nitro-benzenesulfonyl chloride (23.96 g, 0.10 mol) as per theprocedure of example 1. ¹H-NMR (500 MHz; DMSO-d₆) δ 8.11˜8.06 (m, 2H),7.78 (m, 1H), 2.94 (t, 2H), 2.67 (t, 2H), 1.89 (s, 3H).

Example 4 N-(2-Aminoethyl)-2-nitro-4-trifluoromethyl-benzenesulfonamideacetic acid salt

[0199] The title compound (23.62 g, 63%) was synthesized from2-nitro-4-trifluoromethyl-benzenesulfonyl chloride (28.96 g, 0.10 mol)as per the procedure of example 1. ¹H-NMR (500 MHz; DMSO-d₆) δ 8.56 (s,1H), 8.28 (d, 1H), 8.24 (d, 1H), 2.92 (t, 2H), 2.65 (t, 2H), 1.87 (s,3H).

Example 5 N-(2-Aminoethyl)-4-nitro-benzenesulfonamide acetic acid salt

[0200] The title compound (2.40 g, 78%) was synthesized from4-nitro-benzenesulfonyl chloride 2.22 g, 10 mmol) as per the procedureof example 1.

Example 6 N-(2-Aminoethyl)-2-chloro-4-nitro-benzenesulfonamide aceticacid salt

[0201] The title compound (2.38 g, 70%) was synthesized from4-chloro-2-nitro-benzenesulfonyl chloride (2.56 g, 0.1 mol) as per theprocedure of example 1.

Example 7 N-[2-(2-Nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester

[0202] To a solution of N-(2-aminoethyl)-2-nitro-benzenesulfonamideacetic acid salt (15.15 g, 49.6 mmol) and triethylamine (15.16 g, 0.15mol) in dichloromethane (100 mL) was added ethyl bromoacetate (16.7 g0.100 mol) with stirring at room temperature. After additional stirringfor 1h, the reaction mixture was washed with brine (100 mL). The organiclayer was dried over magnesium sulfate and filtered. The filtrate wasevaporated in vacuo and the residue was triturated in ethyl ether (100mL) to afford the titled compound as a white solid (8.1 g, 49%). ¹H-NMR(500 MHz ; DMSO-d₆) δ 8.02˜7.96 (m, 2H), 7.87˜7.84 (m, 2H), 4.05 (q,2H), 3.23 (s, 2H), 2.95 (t, 2H), 2.57 (t, 2H) 1.16 (t, 3H).

Example 8 N-[2-(4-Chloro-2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0203] The title compound (8.3 g, 46%) was synthesized fromN-(2-aminoethyl)-4-chloro-2-nitro-benzenesulfonamide acetic acid salt(16.79 g, 49.4 mmol) as per the procedure of example 7. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.26 (s, 1H), 8.01 (d, 1H), 7.96 (d, 1H), 4.06 (q, 2H), 3.23(s, 2H), 2.95 (t, 2H), 2.57 (t, 2H), 1.16 (t, 3H).

Example 9 N-[2-(4-Fluoro-2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0204] The title compound (7.7 g, 42%) was synthesized fromN-(2-aminoethyl)-4-fluoro-2-nitro-benzenesulfonamide, acetic acid salt(16.79 g, 51.9 mmol) as per the procedure of example 7. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.08 (m, 2H), 7.76 (m, 1H), 4.06 (q, 2H), 3.24 (s, 2H), 2.95(t, 2H), 2.57 (t, 2H), 1.16 (t, 3H).

Example 10N-[2-(2-Nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0205] The title compound (9.23 g, 46%) was synthesized fromN-(2-aminoethyl)-2-nitro-4-trifluoromethyl-benzenesulfonamide aceticacid salt (18.76 g, 50.3 mmol) as per the procedure of example 7. ¹-NMR(500 MHz; DMSO-d₆) δ 8.55 (s, 1H), 8.27 d, 1H), 8.22 (d, 1H), 4.05 (q,2H), 3.22 (s, 2H), 2.99 (t, 2H), 2.57 (t, 2H), 1.16 (t, 3H).

Example 11 N-[2-(4-Nitro-benzenesulfonylamino)-ethyl]-glycine ethylester

[0206] The title compound (826 mg, 50%) was synthesized fromN-(2-aminoethyl)-4-nitro-benzenesulfonamide acetic acid salt (1.52 g,5.2 mmol) as per the procedure of example 7. ¹-NMR (500 MHz ; DMSO-d₆) δ8.41 (d, 2H), 8.04 (d, 2H), 4.05 (q, 2H), 3.23 (s, 2H), 2.86 (t, 2H),2.54 (t, 2H), 1.16 (t, 3H).

Example 12 N-[2-(2-Chloro-4-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0207] The title compound (885 mg, 48%) was synthesized fromN-(2-aminoethyl)-2-chloro-4-nitro-benzenesulfonamide acetic acid salt(1.68 g, 5 mmol) as per the procedure of example 7. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.43 (d, 1H), 8.32 (dd, 1H), 8.22 (d, 11H), 4.04 (q, 2H),3.20 (s, 2H), 2.96 (t, 2H), 2.54 (t, 2H), 1.16 (t, 3H).

Example 13 N-[2-(4-Methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0208] To a solution of N-(2-aminoethyl)-glycine ethyl ester 2HCl (1.10g, 5.0 mmol), prepared as described by Will (D. W. Will et al.,Tetrahedron, 1995, 51, 12069-12802.), in dichloromethane (50 mL) wasslowly added triethylamine (2.02 g, 20 mmol) at room temperature. Then4-methyl-2-nitro-benzenesulfonyl chloride (1.19 g 5.0 mmol) indichloromethane (10 mL) was added to the reaction mixture at roomtemperature for 5 min. The resulting reaction mixture was stirred foradditional 2 h. at room temperature and washed with water (30 mL). Theorganic layer was dried over MgSO₄ and filtered. The filtrate wasevaporated in vacuo to give desired product (1.60 g, 92%) as a solid.¹H-NMR (500 MHz; DMSO-d₆) δ 7.87 (d, 1H), 7.81 (s, 1H), 7.66 (d, 1H),4.06 (q, 2H), 3.24 (s, 2H), 2.91 (t, 2H), 2.57 (t, 2H), 2.44 (s, 3H),1.17 (t, 3H).

Example 14N-[2-(4-Chloro-6-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0209] The title compound (1.72 g, 90%) was synthesized by the reactionof N-(2-aminoethyl)-glycine, ethyl ester 2HCI (1.10 g, 5.0 mmol) with4-chloro-6-methyl-2-nitro-benzenesulfonyl chloride (1.36 g, 5.0 mmol) asper the procedure of example 13.

Example 15N-[2-(4,6-Dichloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethylester

[0210] The title compound (1.83 g, 87%) was synthesized by the reactionof N-(2-aminoethyl)-glycine, ethyl ester 2HCl (1.10 g, 5.0 mmol) with4,6-dichloro-2-nitro-benzenesulfonyl chloride (1.46 g, 5.0 mmol) as perthe procedure of example 13.

EXAMPLE 16 N-[2-(2-Nitro-benzenesulfonylamino)-ethyl]-alanine ethylester

[0211] N-(2-Aminoethyl)-alanine ethyl ester 2HCl (1.165 g, 5.0 mmol),prepared as described by Puschl (A. Puschl et al., Tetrahedron, 1998,39, 4707-4710.), was reacted with 2-nitro-benzenesulfonyl chloride (1.11g 5.0 mmol) as per the procedure of example 13 to give the titlecompound (1.64 g, 95%). ¹H-NMR (500 MHz; DMSO-d₆) δ 8.02˜7.96 (m, 2H),7.86 (m, 2H), 4.05 (q, 2H), 3.16 (q, 1H), 2.94 (t, 2H), 2.58 (m, 1H),2.43 (m, 1H), 1.17 (t, 3H), 1.09 (d, 3H).

Example 17 N-[2-(2-Nitro-benzenesulfonylamino)-ethyl]-phenylglycinemethyl ester

[0212] The title compound (716 mg, 91%) was synthesized by the reactionof N-(2-aminoethyl)-phenylglycine, methyl ester 2HCl (416 mg, 2 mmol)2-nitro-benzenesulfonyl chloride (592 mg, 2.0 mmol) as per the procedureof example 13. ¹H-NMR (500 MHz; DMSO-d₆) δ 8.00˜7.94 (m, 2H), 7.87˜7.81(m, 2H), 7.34˜7.26 (m, 5H), 4.32 (s, 1H), 3.57 (s, 3H), 2.98 (t, 2H),2.54˜2.41 (m, 2H).

Example 18N-t-Butyloxycarbonyl-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0213] To a solution ofN-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester (6.66 g,20.1 mmol) in tetrahydrofuran (50 mL) was added a solution of LiOH( 1.64g, 40 mmol) dissolved in water (30 mL). After stirring for 1 hour atroom temperature, di-t-butyl dicarbonate (6.55 g, 30 mmol) was added tothe reaction mixture. The resulting reaction mixture was stirred for 30min, and then a solution of LiOH (0.82 g, 0.02 mol) in water (15 mL) wasadded. After completion of the reaction by TLC, the precipitate wasremoved by filtration and tetrahydrofurane was evaporated in vacuo. Theresidual solution was washed with ethyl ether (100 mL). The aqueouslayer was acidified to pH=3 by adding 2N HCl and extracted withdichloromethane (100 mL). The organic layer was dried over MgSO₄ andfiltered. The filtrate was concentrated in vacuo to afford the desiredproduct (7.9 g, 98%). ¹H-NMR (500 MHz; DMSO-d₆) δ 8.00 (m, 2H), 7.86 (m,2H), 3.80 (s, 1H), 3.76 (s, 1H), 3.24 (m, 2H), 3.05 (m, 2H), 1.35 (s,4.5H), 1.31 (s, 4.5H).

Example 19N-t-Butyloxycarbonyl-N-[2-(4-chloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0214] The title compound (8.1 g, 92%) was synthesized fromN-[2-(4-chloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester(7.35 g, 20 mmol) as per the procedure of example 18. ¹H-NMR(500 MHz;DMSO-d₆) δ 8.28 (s, 1H), 7.97 (s, 2H), 3.78 (s, 1H), 3.76 (s, 1H), 3.24(m, 2H), 3.06 (m, 2H), 1.35 (s, 4.5H) 1.31 (s, 4.5H).

EXAMPLE 20N-t-Butyloxycarbonyl-N-[2-(4-fluoro-2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0215] The title compound (7.9 g, 89%) was synthesized fromN-[2-(4-fluoro-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester(7.35 g, 21 mmol) as per the procedure of example 18. ¹H-NMR (500 MHz ;DMSO-d₆) δ 8.14˜8.02 (m, 2H), 7.76 (m, 1H), 3.82 (s, 1H), 3.77 (s, 1H),3.24 (m, 2H), 3.05 (m, 2H), 1.36 (s, 4.5H), 1.32 (s, 4.5H).

Example 21N-t-Butyloxycarbonyl-N-[2-(2-nitro-4-trifluoromethyl-benzenesulfonylamiino)-ethyl]-glycine

[0216] The title compound (8.62 g, 91%) was synthesized fromN-[2-(2-nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-glycineethyl ester (8.00 g, 20 mmol) as per the procedure of example 18. ¹H-NMR(500 MHz; DMSO-d₆) δ 8.57 (d, 1H), 8.41 (t, 0.5H), 8.35 (t, 0.5H), 8.19(dd, 1H), 3.82 (s, 1H), 3.75 (s, 1H), 3.24 (t, 2H), 3.09 (m, 1H), 1.34(s, 4.5H), 1.30 (s, 4.5H).

Example 22N-t-Butyloxycarbonyl-[2-(4-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0217] The title compound (7.80 g, 93%) was synthesized fromN-[2-(4-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester(6.91 g, 0.02 mol) as per the procedure of example 18. ¹H-NMR (500 MHz;DMSO-d₆) δ 7.99˜7.93 (m, 1H), 7.85 (dd, 1H), 7.82 (s, 1H), 7.67 (d, 1H),3.81 (s, 1H), 3.76 (s, 1H), 3.23 (m, 2H), 3.05 (m, 2H), 2.44 (s, 3H),1.36 (s, 4.5H), 1.31 (s, 4.5H).

Example 23N-t-Butyloxycarbonyl-N-[2-(4-chloro-6-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0218] The title compound (413 mg, 91%) was synthesized fromN-[2-(4-chloro-6-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester (380 mg, 0.01 mol) as per the procedure of example 18.

Example 24N-t-Butyloxycarbonyl-N-[2-(4,6-dichloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0219] The title compound (417 mg, 88%) was synthesized fromN-[2-(4,6-dichloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethylester (400 mg, 1.0 mmol) as per the procedure of example 18.

Example 25N-t-Butyloxycarbonyl-N-[2-(4-nitro-benzenesulfonylamino)-ethyl]-glycine

[0220] The title compound (362 mg, 90%) was synthesized fromN-[2-(4-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester (331 mg,1.0 mmol) as per the procedure of example 18. ¹H-NMR (500 MHz; DMSO-d₆)δ 8.42 (dd, 2H), 8.03 (d, 2H), 8.01 (br, 1H), 3.80 (s, 1H), 3.22 (m,2H), 2.96 (mn, 2H), 1.34 (s, 4.5H), 1.31 (s, 4.5H).

Example 26N-t-Butyloxycarbonyl-N-[2-(2-chloro-4-nitro-benzenesulfonylamino)-ethyl]-glycine

[0221] The title compound (371 mg, 85%) was synthesized fromN-[2-(2-chloro-4-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester(366 mg, 20 mmol) as per the procedure of example 18. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.44 (q, 1H), 8.35˜8.27 (m, 2H), 8.20 (d, 1H), 3.81 (s, 1H),3.73 (s, 1H), 3.22 (t, 2H), 3.06 (m, 2H), 1.35 (s, 4.5H), 1.30 (s,4.5H).

Example 27N-(t-Butyloxycarbonyl)-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-alanine

[0222] To a solution ofN-[2-(2-nitro-benzenesulfonylamino-ethyl)-alanine ethyl ester(1.04 g, 3mmol) in tetrahydrofuran (10 mL) was added a solution of LiOH (252 mg, 6mmol) dissolved in water (10 mL). After stirring for 1 hour at roomtemperature, di-t-butyl dicarbonate (983 mg, 4.5 mmol) was added to thereaction mixture. The resulting reaction mixture was stirred for 5 h,and then a solution of LiOH (126 mg, 3 mmol) in water (15 mL) was added.After completion of the reaction by TLC, the precipitate was removed byfiltration and tetrahydrofurane was evaporated in vacuo. The residualsolution was washed with ethyl ether (20 mL). The aqueous layer wasacidified to pH=3 by adding 2N HCl and extracted with dichloromethane(30 mL). The organic layer was dried over MgSO₄ and filtered. Thefiltrate was concentrated in vacuo to afford the desired product (1.16g, 93%). ¹H-NMR (500 MHz; DMSO-d₆) δ 7.99 (in, 2H), 7.87 (m, 2H), 4.31(q, 0.4H), 4.03 (q, 0.6H), 3.37˜3.19 (m, 2H), 3.10˜2.95 (in, 2H), 1.35(s, 4.5H), 1.33 (s, 4.5H), 1.30 (d, 1.5H), 1.29 (d, 1.5H).

Example 28N-(t-Butyloxycarbonyl)-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-phenylglycine

[0223] The title compound (583 mg, 81%) was synthesized fromN-[2-(2-nitro-benzenesulfonylamino)-ethyl]-phenylglycine methyl ester(590 mg, 1.5 mmol) as per the procedure of example 27. ¹H-NMR (500 MHz;DMSO-d₆) δ 7.97˜7.82 (m, 4H), 7.35˜7.30 (m, 3H), 7.23˜7.21 (m, 2H), 5.58(s, 0.6H), 5.40 (s, 0.4H), 3.20˜2.85 (m, 4H), 1.46 (s, 4.5H), 1.37 (s,4.5H).

Example 29 1-(2-Nitrobenzenesulfonyl)-piperazin-2-one HCl salt

[0224] To a solution ofN-(t-butoxycarbonyl)-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine(8.06 g, 20 mmol) in dichloromethane (100 ml) was added dicyclohexylcarbodiimide (5.16 g, 25 mmol). After stirring for 2 hours at roomtemperature, the precipitate was removed by filtration. The filtrate wasconcentrated and the residue was dissolved in ethyl actate (50 mL) andcooled to 0° C. The precipitate solid was removed and to the filtratewas added 2N-HCl in ethyl acetate (100 mL). The mixture was stirred foradditional 10 h at ambient temperature. The precipitate product wasfiltered off, washed with ethyl acetate (500 mL), and dried in vacuo toafford the title compound as a white solid 5.39 g (79%). ¹H-NMR (500MHz; DMSO-d₆) δ 9.95 (br.s, 2H), 8.38 (d, 1H), 8.04˜7.95 (m, 2H), 4.09 (dd,2H), 3.96 (s, 2H), 3.56 (dd, 2H).

Example 30 1-(4-Chloro-2-nitrobenzenesulfonyl)-piperazin-2-one HCl salt

[0225] The title compound (4.3 g, 67%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(4-chloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine(7.92 g, 18 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 10.01 (br.s, 211), 8.42 (d, 1H), 8.35 (dd, 1H), 8.06 (dd,1H), 4.07 (t, 2H), 3.95 (s, 2H), 3.55 (t, 2H).

EXAMPLE 31 1-(4-Fluoro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt

[0226] The title compound (4.2 g, 69%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(4-fluoro-2-nitro-benzenesulfonylamino)-ethyl]-glycine(7.61 g, 18 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 9.91 (br.s, 2H), 8.43 (m, 1H), 8.27 (m, 1H), 7.87 (m, 1H),4.06 (dd, 2H), 3.96 (s, 2H), 3.55 (dd, 2H).

Example 32 1-(2-Nitro-4-trifluoromethyl-benzenesulfonyl)-piperazin-2-oneHCl salt

[0227] The title compound (2.55 g, 65%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(2-nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-glycine(4.71 g, 10 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 10.16 (br.s, 2H), 8.71 (s, 1H), 8.60 (d, 1H), 8.35 (d, 1H),4.13 (dd, 2H), 3.96 (s, 2H), 3.57 (dd, 2H).

Example 33 1-(4-Methyl-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt

[0228] The title compound (2.40 g, 71%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(4-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycine(4.19 g, 10 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 9.87 (br.s, 1H), 8.23 (d, 1H), 7.98 (s, 1H), 7.77 (d, 1H),4.06 (dd, 2H), 3.95 (s, 2H), 3.55 (dd, 2H), 2.49 (s, 3H).

Example 34 1-(4-Chloro-6-methyl-2-nitro-benzenesulfonyl)-piperazin-2-oneHCl salt

[0229] The title compound (2.53 g, 68%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(4-chloro-6-methyl-2-nitro-benzenesulfonylamino)-ethyl]-glycine(4.54 g, 10 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 10.02 (br.s, 2H), 8.20 (s, 1H), 7.96 (s, 1H), 4.04 (dd, 2H),3.96 (s, 2H), 3.53 (dd, 2H), 2.67 (s, 3H).

Example 35 1-(4,6-Dichloro-2-nitro-benzenesulfonyl)-piperazin-2-one HClsalt

[0230] The title compound (2.51 g, 64%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(4,6-dichloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine(4.74 g, 10 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 10.06 (br.s, 2H), 8.43 (s, 1H), 8.32 (s, 1H), 4.16 (t, 2H),3.99 (s, 2H), 3.50 (t, 2H).

Example 36 1-(4-Nitrobenzenesulfonyl)-piperazin-2-one HCl salt

[0231] The title compound (208 mg, 65%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(4-nitro-benzenesulfonylamino)-ethyl]-glycine(403 mg, 1 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 9.92 (br.s, 2H), 8.43 (d, 2H), 8.29 (d, 2H), 4.17 (dd, 2H),4.03 (s, 2H), 3.54 (dd, 2H).

Example 37 1-(2-Chloro-4-nitrobenzenesulfonyl)-piperazin-2-one HCl salt

[0232] The title compound (239 mg, 67%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(2-chloro-4-nitro-benzenesulfonylamino)-ethyl]-glycine(437 mg, 1 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 10.28 (br.s, 2H), 8.53 (d, 1H), 8.45 (d, 1H), 8.39 (dd, 1H),4.25 (dd, 2H), 3.94 (s, 2H), 3.54 (dd, 2H).

EXAMPLE 38 1-(2-Nitro-benzenesulfonyl)-3-methyl-piperazin-2-one HCl salt

[0233] The title compound (450 mg, 67%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-alanine(835 mg, 2 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.38 (d, 1H), 8.14 (d, 1H), 8.03 (dd, 1H), 7.98 (dd, 1H),4.30 (m, 1H), 4.13 (m, 2H), 3.70 (m, 1H), 3.51 (m, 1H), 1.39 (d, 3H).

Example 39 1-(2-Nitro-benzenesulfonyl)-3-phenyl-piperazin-2-one HCl salt

[0234] The title compound (247 mg, 63%) was synthesized fromN-(t-butyloxycarbonyl)-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-phenylglycine(480 mg, 1 mmol) as per the procedure of example 29. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.35 (d, 1H), 8.15 (d, 1H), 8.02 (t, 1H), 7.94 (t, 1H), 7.40(m, 5H), 5.47 (br s, 1H), 4.24 (m, 2H), 3.69 (m, 1H), 3.60 (m, 1H).

Example 40 (2-Amino-6-iodopurin-9-yl)-acetic acid ethyl ester

[0235] To a solution of 2-amino-6-iodo-purine (78.3 g, 0.3 mol) in DMF(1960 mL) was added ethyl bromoacetate (55.1 g, 0.33 mol) and potassiumcarbonate (82.9 g, 0.6 mol). The resulting reaction mixture was stirredfor 12 h at room temperature. The reaction mixture was concentrated tosmall volume (about 150 mL) in vacuo and the residue was dissolved inwater The solid was filtered off, washed with water and ethyl ether, anddried in vacuo to give the titled compound (98.4 g, 95%). ¹H-NMR (500MHz ; DMSO-d₆) δ 8.06 (s, 1H), 6.90 (br.s, 2H), 4.94 (s, 2H), 4.17 (q,2H), 1.22 (t, 3H).

Example 41 [2-(Benzhydryloxycarbonyl)-amino-6-iodopurine-9-yl]-aceticacid ethyl ester

[0236] To a solution of (2-amino-6-iodopurine-9-yl)-acetic acid ethylester (13.9 g, 40 mmol) in THF (280 mL) was added triphosgene (5.34 g,18 mmol) at 0° C. After stirring for additional 5 min,N,N-diisopropylethylamine (24.4 mL) was slowly added and the reactionmixture was stirred for 30 min at 0° C. Then benzhydrol was added andthe resulting reaction mixture was allowed to warm to room temperatureand stirred for additional 13 h. The reaction mixture was neutralized byaddition of 1N HCl solution and saturated with sodium chloride andsodium thiosulfate. The organic layer was separated and the aqueouslayer was extracted with ethyl acetate (200 mL×3). The combined organiclayer was washed with brine (200 mL×2), dried over sodium sulfate, andfiltered. The filtrate was evaporated in reduced pressure and theresidue was purified by column chromatography to afford the titledcompound (15.15g, 68%). ¹H-NMR (500 MHz; DMSO-d₆) δ 10.88 (bs, 1H), 8.45(s, 1H), 7.60˜7.20 (m, 10H), 6.79 (s, 1H), 5.08 (s, 2H), 4.15 (q, 2H),1.17 (t, 3H).

EXAMPLE 42 [2-N-(Benzhydryloxycarbonyl)-guanin-9-yl]-acetic acid

[0237] To a suspension of 60% NaH (5.04 g, 126 mmol) in THF (110 mL) wasslowly added 3-hydroxypropionitrile for a period of 10 min at 0° C. andthe mixture was stirred for additional 12 min. To the resulting reactionmixture was slowly added[2-(benzhydryloxycarbonyl)-amino-6-iodo-purine-9-yl]-acetic acid (11.12g, 21 mmol) portionwise in an ice bath. After the addition wascompleted, the ice bath was removed and stirring continued foradditional 3.5 h. Then the reaction mixture was acidified by addition of20% aqueous solution of citric acid and saturated with sodium chloride.The organic layer was separated and the aqueous layer was extracted withTHF (300 mL×2). The combined organic layer was dried over sodium sulfateand filtered. The solvent was removed in vacuo and the residue wasrecrystallized in ethyl alcohol. The solid was filtered off, washed withcold ethyl acohol, and dried in vacuo to give the desired product (9.00g).

Example 43 [2-(Benzyloxycarbonyl)-amino-6-iodo-purine-9-yl]-acetic acidethyl ester

[0238] The title compound (12.42 g, 64.4%) was synthesized by thereaction of (2-amino-6-iodopurine-9-yl)-acetic acid ethyl ester (13.9 g,40 mmol) and phosgene followed by benzyl alcohol treatment as per theprocedure of example 41. ¹H-NMR (500 MHz; DMSO-d₆) δ 10.83 (bs, 1H),8.48 (s, 1H), 7.50˜7.30 (m, 5H), 5.18 (s, 2H), 5.11 (s, 2H), 4.19 (q,2H), 1.21 (t, 3H).

EXAMPLE 44 [2-(Benzyloxycarbonyl)-amino-6-iodopurin-9-yl]-acetic acid

[0239] To a suspension of[2-(benzyloxycarbonyl)-amino-6-iodo-purine-9-yl]-acetic acid ethyl ester(10.02 g, 20.8 mmol) in tetrahydrofuran (50 mL) and water (50 mL) wasadded lithium hydroxide hydrate (2.83 g, 20.8 mmol) at 10° C. Theresulting reaction mixture was stirred for 30 min. Then the mixture wasacidified to pH=3 by adding 1N HCl. The precipitated solid was filteredoff, washed with water and ethyl ether, and dried in vacuo to give thetitle compound (9.81 g). ¹H-NMR (500 MHz; DMSO-d₆) δ 10.75 (s, 1H), 8.44(s, 1H), 7.50˜7.30 (m, 5H), 5.17 (s, 2H), 4.97 (s, 2H).

Example 45 [2-N-(Benzyloxycarbonyl)-guanin-9-yl]-acetic acid

[0240] The title compound (5.44 g, 79.2%) was synthesized from[2-(benzyloxycarbonyl)-amino-6-iodopurin-9-yl]-acetic acid (9.06 g, 20mmol) as per the procedure of example 42. ¹H NMR (DMSO-d6) δ 11.54 (s,1H), 11.37 (s, 1H), 7.94 (s, 1H), 7.46˜7.33 (m, 5H), 5.28 (s, 2H), 4.87(s, 2H).

EXAMPLE 46N-[2-(2-Nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester

[0241] To the mixture of N-[2-(2-nitro-sulfonylamino)-ethyl]-glycineethyl ester (1.67 g, 5 mmol), (thymin-1-yl)-acetic acid (0.92 g, 5mmol), and PyBOP (3.12 g, 6 mmol) in DMF (15 mL) was addedN,N-diisopropylethylamine (1.31 mL, 7.5 mmol) at ambient temperature.The resulting reaction mixture was stirred for 7 h at the sametemperature and the solvent was removed in vacuo. The residue wasdissolved in ethyl acetate (50 mL) and washed with 1N HCl aqueoussolution, saturated sodium bicarbonate solution, and brine. The organiclayer was dried over magnesium sulfate and filtered. The filtrate wasconcentrated and the residue was triturated with ethyl alcohol. Theresulting solid was filtered off and dried in vacuo to give the titlecompound as a white solid (2.35 g, 93%). ¹H-NMR (500 MHz; DMSO-d₆) δ11.29 (s, 0.6H), 11.26 (s, 0.4H), 8.25˜7.87 (m, 5H), 7.30 (s, 0.6H),7.23 (s, 0.4H), 4.64 (s, 1.2H), 4.46 (s, 0.8H), 4.29 (s, 0.8H), 4.16 (q,0.8H), 4.07 (q, 1.2H), 4.00 (s, 1.2H), 3.49 (t, 1.2H), 3.21 (q, 1.2H),3.12˜3.01 (m, 1.6H), 1.75 (s, 3H), 1.22 (t, 1.2H), 1.17 (t, 1.8H).

EXAMPLE 47N-[2-(4-Chloro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester

[0242] The title compound (2.46 g, 92%) was synthesized by the reactionof N-[2-(4-chloro-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethylester (1.84 g, 5 mmol) and (thymin-1-yl)-acetic acid (0.92 g, 5 mmol) asper the procedure of example 46. ¹H-NMR (500 MHz; DMSO-d₆) δ 11.31 (s,0.6H), 11.28 (s, 0.4H), 8.31˜7.95 (m, 4H), 7.31 (s, 0.6H), 7.23 (s,0.4H), 4.64 (s, 1.2H), 4.46 (s, 0.8H), 4.28 (s, 0.8H), 4.16 (q. 0.8H),4.07 (q. 1.2H), 4.00 (s, 1.2H), 3.49 (t, 1.2H), 3.22 (q, 1.2H),3.12˜3.01 (m, 1.6H), 1.74 (s, 3H), 1.22 (t, 1.2H), 1.17 (t, 1.8H).

EXAMPLE 48N-[2-(4-Fluoro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester

[0243] The title compound (2.35 g, 91%) was synthesized by the reactionof N-[2-(4-fluoro-2-nitro-benzenesulfonylamino)-ethyl]-glycine ethylester (1.76 g, 5 mmol) and (thymin-1-yl)-acetic acid (0.92 g, 5 mmol) asper the procedure of example 46. ¹H-NMR (500 MHz; DMSO-d₆) δ 11.30 (s,0.6H), 11.27 (s, 0.4H), 8.28˜8.05 (m, 2.8H), 7.78 (m, 1.2H), 7.31 (s,0.6H), 7.23 (s, 0.4H), 4.65 (s, 1.2H), 4.47 (s, 0.8H), 4.30 (s, 0.8H),4.16 (q. 0.8H), 4.07 (q. 1.2H), 4.00 (s, 1.2H), 3.49 (t, 1.2H), 3.22 (q,1.2H), 3.12˜3.01 (m, 1.6H), 1.75 (s, 3H), 1.23 (t, 1.2H), 1.18 (t,1.88H).

EXAMPLE 49N-[2-(2-Nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester

[0244] The title compound (2.69 g, 95%) was synthesized by the reactionof N-[2-(2-Nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-glycineethyl ester (2.00 g, 5 mmol) and (thymin-1-yl)-acetic acid (0.92 g, 5mmol) as per the procedure of example 46. ¹H-NMR (500 MHz; DMSO-d₆)δ11.31 (s, 0.55H), 11.28 (s, 0.45H), 8.57 (s, 0.55H), 8.54 (s, 0.45H),8.50 (t, 0.55H), 8.38 (t, 0.45H), 8.29˜8.18 (m, 2H), 7.31 (s, 0.55H),7.22 (s, 0.45H), 4.64 (s, 1.1H), 4.46 (s, 0.9H), 4.21 (s, 0.9H), 4.16(q. 0.9H), 4.07 (q. 1.1H), 3.95 (s, 1.1H), 3.49 (t, 1.1H), 3.37 (q,1.1H), 3.25 (t, 0.9H), 3.09 (t, 0.9H), 1.75 (s, 3H), 1.23 (t, 1.35H),1.18 (t, 1.65H).

Example 50 N-{[4-N-(Benzhydryloxycarbonyl)-cytosin- 1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine ethylester

[0245] The title compound (2.45 g, 88%) was synthesized by the reactionof N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine ethyl ester (1.33g, 4 mmol) and 2-[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetic acid(1.51 g, 4 mmol) as per the procedure of example 46. ¹H-NMR (500 MHz;DMSO-d₆) δ 10.99 (s, 0.6H,), 10.98 (s, 0.4H), 8.30˜7.80 (m, 5H),7.50˜7.25 (m, 10H), 6.94 (t, 1H), 6.79 (s, 1H), 4.79 (s, 1.2H), 4.61 (s,0.8H), 4.33 (s, 0.8H), 4.15 (q, 0.8H), 4.05 (q, 1.2H), 4.01 (s, 1.2H),,3.52 (t, 1.2H), 3.36 (t, 0.8H), 3.24 (q, 1.2H), 3.02 (q, 0.8H), 1.23 (t,1.2H), 1.15 (t, 1.8H).

Example 51N-{[4-(Benzyloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester

[0246] The title compound (yield: 78%) was synthesized as per theprocedure of example 46. ¹H-NMR (500 MHz; DMSO-d₆) δ 8.15˜8.06 (mn, 1H),7.84˜7.57 (m, 5H), 7.38˜7.32 (m, 6H), 7.14 (t, 0.7H), 6.44 (t, 0.3H),5.20 (s, 2H), 4.93 (s, 1.4H), 4.55 (s, 0.6H), 4.36 (s, 0.6H), 4.26 (q,0.6H), 4.17 (q, 1.4H) 4.03 (s, 1.4H), 3.71 (t, 1.4H), 3.57 (t, 0.6H),3.39 (q, 1.4H), 3.30 (q, 0.6H), 1.31 (t, 0.9H), 1.24 (t, 2.1 H).

EXAMPLE 52N-[2-(2-Nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine

[0247] To a solution ofN-[2-(2-Nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester (1.00 g, 2.0 mmol) in THF (10 ml,) was added a solution oflithium hydroxide (210 mg, 5 mmol) in water (10 mL) at 10° C. Afterstirring for 1.5 h, the reaction mixture was acidified to pH=23 byadding 1N HCl solution. The precipitated solid was filtered off, washedwith water, and dried in vacuo to give the tilted product as a whitesolid (896 mg, 95%). ¹H-NMR (500 MHz; DMSO-d₆) δ 11.30 (s, 0.6H), 11.27(s, 0.4H), 8.22˜7.85 (m, 5H), 7.31 (d, 0.6H), 7.23 (d, 0.4H), 4.63 (s,1.2H), 4.45 (s, 0.8H), 4.19 (s, 0.8H), 4.16 (q. 0.8H), 3.93 (s, 1.2H),3.47 (t, 1.2H), 3.34 (t, 0.8H), 3.20 (q, 1.2H), 3.04 (q. 0.8H), 1.75 (s,3H).

Example 53 N-[2-(4-Chloro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine

[0248] The title compound (1.40 g, 93%) was synthesized fromN-[2-(4-chloro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester (1.60 g, 3.0 mmol) as per the procedure of example 52.¹H-NMR (500 MHz; DMSO-d₆) δ 11.31 (s, 0.55H), 11.28 (s, 0.45H),8.33˜7.95 (m, 4H), 7.31 (s, 0.55H), 7.23 (s, 0.45H), 4.63 (s, 1.1H),4.44 (s, 0.9H), 4.20 (s, 0.9H), 4.00 (s, 1.1H), 3.47 (t, 1.1H), 3.34 (t,0.9H), 3.19 (q, 1.1H), 3.04 (q, 0.9H), 1.75 (s, 3H).

Example 54N-[2-(4-Fluoro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine

[0249] The title compound (0.92 g, 94%) was synthesized fromN-[2-(4-fluoro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester (1.03 g, 2.0 mmol) as per the procedure of example 52.¹H-NMR (500 MHz; DMSO-d₆) 11.30 (s, 0.6H), 11.28 (s, 0.4H), 8.29˜8.05(m, 2.8H), 7.78 (m, 1.2H), 7.31 (s, 0.6H), 7.23 (s, 0.4H), 4.63 (s,1.2H), 4.44 (s, 0.8H), 4.20 (s, 0.8H), 3.94 (s, 1.2H), 3.46 (t, 1.2H),3.34 (t, 0.8H), 3.20 (q, 1.2H), 3.04 (q. 0.8H), 1.75 (s, 3H).

Example 55N-[2-(2-Nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-N-[(thymin-1 -yl)-acetyl]-glycine

[0250] The title compound (1.02 g, 95%) was synthesized fromN-[2-(2-nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester (1.14 g, 2.0 mmol) as per the procedure of example 52.¹H-NMR (500 MHz; DMSO-d₆) δ 11.31 (s, 0.55H), 11.28 (s, 0.45H), 8.58 (s,0.55H), 8.54 (s, 0.45H), 8.51 (t, 0.55H), 8.38 (t, 0.45H), 8.29˜8.18 (m,2H), 7.31 (s, 0.55H), 7.22 (s, 0.45H), 4.63 (s, 1.11H), 4.45 (s, 0.9H),4.20 (s, 0.9H), 3.94 (s, 1.11H), 3.48 (t, 1.1H), 3.37 (t, 0.9H), 3,24(q, 1.11H), 3.09 (q, 0.9H), 1.75 (s, 3H).

Example 56N-{[4-N-(Benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0251] To a solution ofN-{[4-N-(Benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylanino)-ethyl]-glycineethyl ester (1.91 g, 2.75 mmol) in THF (10 mL) was added a solution oflithium hydroxide (290 mg, 6.9 mmol) in water (9 mL) at 10° C. Afterstirring for 1.5 h, the reaction mixture was acidified to pH=3˜4 byadding 1N HCl solution. The aqueous layer was saturated with sodiumchloride and organic layer was separated. The aqueous layer wasextracted with THF (15 mL×2). The combined organic layer was dried oversodium sulfate and filtered. The filtrate was concentrated andtriturated with ethyl alcohol to precipitate solid. The precipitatedsolid was filtered off, washed with water, and dried in vacuo to givethe tilted product as a white solid (1.42 g, 78%). ¹H-NMR (500 MHz;DMSO-d₆) δ 8.25˜7.80 (mn, 5H), 7.50˜7.25 (m, 10H), 6.94 (d, 0.6H), 6.92(d, 0.4H), 6,79 (1H, s), 4.78 (s, 1.2H), 4.60 (s, 0.8H), 4.22 (s, 0.8H),3.94 (s, 1.2H), 3.50 (t, 1.2H), 3.35 (t, 0.8H), 3.24 (q, 1.2H), 3.02 (q,0.8H).

Example 57N-{[4-N-(Benzyloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine

[0252] The title compound (yield: 70%) was synthesized fromN-{[4-N-(Benzyloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycineethyl ester as per the procedure of example 56. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.26˜7.83 (m, 5H), 7.44˜7.33 (m, 5H), 7.02 (d, 0.6H), 7.00(d, 0.4H), 5.19 (s, 2H) 4.80 (s, 1.2H), 4.61 (s, 0.8H), 4.23 (s, 0.8H),3.94 (s, 1.2H), 3.50 (t, 1.2H), 3.35 (t, 0.8H), 3.24 (q, 1.2H), 3.02 (q,0.8H).

Example 581-(2-Nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0253] To a solution ofN-[2-(2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine(469 mg, 1.0 mmol) and N-mehtyl morpholine (330 μL, 3 mmol) in THF (10mL) was added isobutylchloroformatre (205 mg, 1.5 mmol) at −20° C. Thereaction mixture was allowed to warm to 0° C. for a period of 1 h. Thenwater (10 mL) was added to the reaction mixture to precipitate solid.The solid was filtered off, washed with water, dried in vacuo to givethe tiltled compound (442 mg, 98%). ¹H-NMR (500 MHz; DMSO-d₆) δ 11.32(s, 0.6H), 11.30 (s, 0.4H), 8.35 (m, 1H), 8.12 (dd, 1H), 8.04˜7.96 (m,2H), 7.35 (s, 0.6H), 7.28 (s, 0.4H), 4.67 (s, 1.2H), 4.59 (s, 0.8H),4.41 (s, 0.8H), 4.27 (s, 1.2H), 4.04 (m, 1.2H), 3.95 (m, 1.2H),3.90˜3.85 (m, 1.6H), 1.75 (s, 3H).

Example 591-(4-Chloro-2-nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one)

[0254] The title compound (933 mg, 96%) was synthesized fromN-[2-(4-chloro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine(1.01 g, 2.0 mmol) as per the procedure of example 58. ¹H-NMR (500 MHz;DMSO-d₆) δ 11.33 (s, 0.6H), 11.31 (s, 0.4H), 8.40 (d, 1H), 8.33 (dd,1H), 8.08 (dd, 1H), 7.34 (s, 0.6H), 7.28 (s, 0.4H), 4.66 (s, 1.2H), 4.59(s, 0.8H), 4.41 (s, 0.8H), 4.26 (s, 1.2H), 4.02 (m, 1.2H), 3.94 (m,1.2H), 3.89˜3.82 (m, 1.6H), 1.74 (s, 3H).

Example 601-(4-Fluoro-2-nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0255] The title compound (451 mg, 96%) was synthesized fromN-[2-(4-fluoro-2-nitro-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine(487 mg, 1.0 mmol) as per the procedure of example 58. ¹H-NMR (500 MHz;DMSO-d₆) δ 11.33 (s, 0.6H), 11.31 (s, 0.4H), 8.41 (m, 1H), 8.25 (dd,1H), 7.88 (m, 1H), 7.34 (s, 0.6H), 7.28 (s, 0.4H), 4.66 (s, 1.2H), 4.59(s, 0.8H), 4.41 (s, 0.8H), 4.26 (s, 1.2H), 4.02 (m, 1.2H), 3.94 (m,1.2H), 3.88˜3.82 (m, 1.6H), 1.74 (s, 3H).

Example 61 1-(2-Nitro-4-trifluoromethyl-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0256] The title compound (493 mg, 95%) was synthesized fromN-[2-(2-nitro-4-trifluoromethyl-benzenesulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine(537 mg, 1.0 mmol) as per the procedure of example 58. ¹H-NMR(500 MHz;DMSO-d₆) δ 11.33 (s, 0.6H), 11.31 (s, 0.4H), 8.69 (s, 1H), 8.56 (dd,1H), 8.39 (dd, 1H), 7.34 (s, 0.6H), 7.28 (s, 0.4H), 4.67 (s, 1.2H), 4.59(s, 0.8H), 4.42 (s, 0.8H), 4.27 (s, 1.2H), 4.05 (m, 1.2H), 3.95 (m,1.2H), 3.91˜3.85 (m, 1.6H), 1.75 (s, 3H).

Example 624-{[4-N-(Benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-1-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0257] To a solution ofN-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycine(1.20 g, 1.8 mmol) in THF (24 mL) was added N-methylmorpholine (0.6 mL,5.42 mmol) and then the mixture was cooled to −20° C. After stirring for5 min at the same temperature, isobutyl chloroformate (0.30 mL, 2.31mmol) was added to the reaction mixture. The resulting mixture wasslowly warmed to 0° C. for 1 h. Then the reaction mixture was evaporatedin vacuo and dissolved in a mixture of ethyl acetate and acetonitrile.The solution was washed with saturated NaCl solution and dried oversodium sulfate and filtered. The filtrate was evaporated in vacuo andtriturated with methanol to precipitate solid. The solid was filteredoff, washed with methanol, and dried in vacuo to give the titledcompound 0.9 g (77%). ¹H-NMR (500 MHz ; DMSO-d₆) δ 8.35 (t, 1H),8.12˜7.95 (m, 3H), 7.90 (d, 0.6H), 7.84 (d, 0.4H), 7.48˜7.28 (m, 10H),6.95 (d, 0.6H), 6.94 (d, 0.4H), 6.79 (s, 1H), 4.82 (s, 1.2H), 4.73 (s,0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.10˜3.95 (m, 2.4H), 3.95˜3.80(m, 1.6H).

Example 634-{[4-N-(Benzyloxycarbonyl)-cytosin-1-yl]-acetyl}-1-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0258] The title compound (yield: 90%) was synthesized fromN-{[4-N-(benzyloxycarbonyl)-cytosin-1-yl]-acetyl}-N-[2-(2-nitro-benzenesulfonylamino)-ethyl]-glycineas per the procedure of example 62. ¹H-NMR (500 MHz; DMSO-d₆) δ 10.76(s, 1H), 8.36 (t, 1H), 8.12 (dd, 1H), 8.02˜7.81 (m, 3H), 7.41˜7.31 (m,5H), 7.03 (s, 0.4H), 7.02 (s, 0.6H), 5.17 (s, 2H), 4.82 (s, 1.2H), 4.74(s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.06 (m, 1.2H), 3.98 (m,1.2H), 3.91 (mn, 0.8H), 3.84 (m, 0.8H).

Example 64 1 -(2-Nitro-benzenesulfonyl)-4-[(thymin- 1-yl)-acetyl]-piperazin-2-one

[0259] To a mixture of 1-(2-nitro-benzenesulfonyl)-piperazin-2-one HClsalt (1.13 g, 3.5 mmol), (thymin-1-yl)-acetic acid (0.64 g, 3.5 mmol),and PyBOP(2.00 g, 3.85 mmol) in DMF (11 mL) was addeedN,N-diisopropylethylamine (0.91 mL) at room temperature. After stirringfor additional 2 h, the reaction mixture was slowly added to a solutionof aqueous ethyl alcohol to precipitate solid. The solid was filteredoff, washed with ethanol and ethyl ether, and dried in vacuo to give thetitled compound (1.50 g, 95%).

Example 65 1 -(4-Chloro-2-nitro-benzenesulfonyl)-4-[(thymin- 1-yl)-acetyl]-piperazin-2-one

[0260] The title compound (1.60 g, 94%) was synthesized by reaction of1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (1.25 g,3.5 mmol) with (thymin-1-yl)-acetic acid (0.64 g, 3.5 mmol) as per theprocedure of example 64.

Example 661-(4-Fluoro-2-nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0261] The title compound (900 mg, 96%) was synthesized by reaction of1-(4-fluoro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (680 mg,2.0 mmol) with (thymin-1-yl)-acetic acid (369 mg, 2.0 mmol) as per theprocedure of example 64.

Example 67 1 -(2-Nitro-4-trifluoromethyl-benzenesulfonyl)-4-[(thymin- 1-yl)-acetyl]-piperazin-2-one

[0262] The title compound (980 mg, 94%) was synthesized by reaction of1-(2-nitro-4-trifluoromethyl-benzenesulfonyl)-piperazin-2-one HCl salt(780 mg, 2 mmol) with (thymin-1-yl)-acetic acid (369 mg, 2 mmol) as perthe procedure of example 64.

Example 681-(4-Methyl-2-nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0263] The title compound (888 mg, 95%) was synthesized by reaction of1-(4-methyl-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (672 mg,2.0 mmol) with (thymin-1-yl)-acetic acid (369 mg, 2.0 mmol) as per theprocedure of example 64. ¹H-NMR (500MHz ; DMSO-d₆) δ 11.33 (s, 0.6H),11.32(s, 0.4H), 8.22(m, 1H), 7.96(s, 1H), 7.78(m, 1H), 7.35(s, 0.6H),7.28(s, 0.4H), 4.66(s, 1.2H), 4.58 (s, 0.8H), 4.39 (s, 0.8H), 4.24 (s,1.211), 4.01 (mn, 1.2H), 3.93 (mn, 1.2H), 3.88˜3.81 (m, 1.6H), 2.49 (s,3H), 1.74 (s, 3H).

Example 69 1-(4-Chloro-6-methyl-2-nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0264] The title compound (913 mg, 91%) was synthesized by reaction of1-(4-chloro-6-methyl-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt(740 mg, 2.0 mmol) with (thymin-1-yl)-acetic acid (368 mg, 2.0 mmol) asper the procedure of example 64. ¹H-NMR (500 MHz; DMSO-d₆) δ 11.34 (s,0.6H), 11.32 (s, 0.4H), 8.19 (s, 1H), 7.95 (s, 1H), 7.34 (s, 0.6H), 7.28(s, 0.4H), 4.67 (s, 1.2H), 4.59 (s, 0.8H), 4.40 (s, 0.8H), 4.26 (s,1.2H), 3.99 (m, 1.2H), 3.92 (m, 1.2H), 3.82 (s, 1.6H), 2.64 (s, 3H),1.74 (s, 3H).

Example 701-(4,6-Dichloro-2-nitro-benzenesulfonyl)-4-[(thymin-1-yl)acetyl]-piperazin-2-one

[0265] The title compound (919 mg, 88%) was synthesized by reaction of1-(4,6-dichloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (781mg, 2.0 mmol) with (thymin-1-yl)-acetic acid (368 mg, 2.0 mmol) as perthe procedure of example 64. ¹H-NMR (500MHz; DMSO-d₆) δ 11.34 (s, 0.6H),11.32 (s, 0.4H), 8.42 (d, 0.4H), 8.41 (d, 0.6H), 8.32 (d, 0.4H), 8.30(d, 0.6H), 7.35 (s, 0.6H), 7.27 (s, 0.4H), 4.69 (s, 1.2H), 4.58 (s,0.8H), 4.42 (s, 0.8H), 4.27 (s, 1.2H), 4.11 (t, 1.2H), 3.97 (t, 0.8H),3.93 (t, 1.2H), 3.82 (t, 0.8H), 1.74 (s, 3H).

Example 711-(4-Nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0266] The title compound (151 mg, 95%) was synthesized by reaction of1-(4-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (113 mg, 0.35 mmol)with (thymin-1-yl)-acetic acid (64 mg, 0.35 mmol) as per the procedureof example 64. ¹H-NMR (500 MHz ; DMSO-d₆) δ 11.31 (s, 0.6H), 11.29 (s,0.4H), 8.44 (d, 2H), 8.29 (d, 2H), 7.34 (s, 0.6H), 7.27 (s, 0.4H), 4.64(s, 1.2H), 4.55 (s, 0.8H), 4.33 (s, 0.8H), 4.18 (s, 1.2H), 4.14 (t,1.2H), 4.00 (t, 0.8H), 3.90 (t, 1.2H), 3.80 (t, 0.8H), 1.74 (s, 3H).

Example 721-(2-Chloro-4-nitro-benzenesulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

[0267] The title compound (159 mg, 94%) was synthesized by reaction of1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (125 mg,0.35 mmol) with (thymin-1-yl)-acetic acid (64 mg, 0.35 mmol) as per theprocedure of example 64. ¹H-NMR (500 MHz; DMSO-d₆) δ 11.32 (s, 0.6H),11.30 (s, 0.4H), 8.53 (d, 1H), 8.47˜8.40 (m, 2H), 7.35 (s, 0.6H), 7.28(s, 0.4H), 4.68 (s, 1.2H), 4.57 (s, 0.8H), 4.38 (s, 0.8H), 4.24 (s,1.2H), 4.18 (t, 1.2H), 4.04 (t, 0.8H), 3.95 (t, 1.2H), 3.84 (t, 0.8H),1.75 (s, 3H).

Example 734-{[6-N-(Benzhydryloxycarbonyl)-adenin-1-yl]acetyl}-1-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0268] To a solution of 1-(2-nitro-benzenesulfonyl)-piperazin-2-one HClsalt (0.76 g, 2.36 mmol),[6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetic acid (1.0 g, 2.47mmol), and PyBOP(1.35 g) in DMF (20 mL) was addedN,N-diisopropylethylamine (0.95 mL) at 5 ° C. After stirring foradditional 2 h, the reaction mixture was diluted with ethyl acetate (200mL) and water (150 mL). The organic layer was separated and washed withwater, 5% aqueous sodium bicarbonate, 10% aqueous citric acid, andbrine. The organic layer was dried over sodium sulfate and concentratedin reduced pressure. The residue was purified by column chromatographyto give the title compound (830 mg, 58%). ¹H NMR (DMSO-d₆) δ 10.95 (s,1H), 8.59 (s, 0.6H), 8.58 (s, 0.4H), 8.40˜8.36 (m, 1H), 8.33 (s, 0.6H),8.31 (s, 0.4H), 8.14 (d, 1H), 8.06˜7.96 (m, 2H), 7.54˜7.27 (m, 10H),6.82 (s, 1H), 5.39 (s, 1.2H), 5.30 (s, 0.8H), 4.56 (s, 0.8H), 4.27 (s,1.2H), 4.12˜4.08 (m, 2.4H), 3.92˜3.86 (m, 1.6H).

Example 744-{[6-N-(Benzhydryloxycarbonyl)-adenin-1-yl]-acetyl}-1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one

[0269] The title compound (810 mg, 56%) was synthesized by reaction of1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (712 mg,2.0 mmol) with [6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetic acid(779 mg, 2.0 mmol) as per the procedure of example 73. ¹H NMR (DMSO-d₆)δ 11.05 (s, 1H), 8.60 (s, 0.6H), 8.59 (s, 0.4H), 8.43˜8.41 (m, 1H),8.37˜8.32 (m, 2H), 8.08 (m, 1H), 7.53˜7.27 (m, 10H), 6.82 (s, 1H), 5.39(s, 1.2H), 5.31 (s, 0.8H), 4.56 (s, 0.8H), 4.27 (s, 1.2H), 4.12˜4.08(br. m, 2.4H), 3.90˜3.85 (m, 1.6H).

Example 754-{[6-N-(Benzhydryloxycarbonyl)-adenin-9-yl]-acetyl}-1-(4-fluoro-2-nitro-benzenesulfonyl)-piperazin-2-one

[0270] The title compound (805 mg, 58%) was synthesized by reaction of1-(4-fluoro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (679 mg,2.0 mmol) with [6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetic acid(807 mg, 2.0 mmol) as per the procedure of example 73. ¹H NMR (DMSO-d₆)δ 10.95 (s, 1H), 8.59 (s, 0.6H), 8.58 (s, 0.4H), 8.47˜8.40 (m, 1H), 8.33(s, 0.6H), 8.30 (s, 0.4H), 8.26 (m, 1H), 7.88 (m, 1H), 7.53˜7.27 (m,10H), 6.82 (s, 1H), 5.38 (s, 1.2H), 5.30 (s, 0.8H), 4.56 (s, 0.8H), 4.27(s, 1.2H), 4.12˜4.06 (m, 2.4H), 3.91˜3.85 (br. m, 1.6H).

Example 764-{[2-N-(Benzhydryloxycarbonyl)-guanin-9-yl]-acetyl}-1-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0271] To a solution of4-[2-N-(bezhydryloxycarbonyl)-guanin-9-yl]-acetic acid (1.89 g, 4.5mmol), 1-(2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (1.45 g, 4.5mmol), and PyBOP (2.81 g, 5.4 mmol) in DMF (13 mL) was addedN,N-diisopropylethylamine (1.96 mL, 13 mmol) at room temperature. Afterstirring for additional stirring for I h, the reaction mixture wasacidified to pH=3˜4 by adding 20% aqueous citric acid solution. Theprecipitated solid was filtered off, washed with brine, andrecrystallized from acetonitrile to give the titled compound (1.15 g,37%). ¹H NMR (DMSO-d₆) δ 11.63 (s, 1H), 11.24 (s, 1H), 8.37 (In, 1H),8.13 (dd, 1H), 8.06˜7.97 (m, 2H), 7.82 (s, 0.6H), 7.78 (s, 0.4H),7.46˜7.28 (m, 10H), 6.86 (s, 1H), 5.15 (s, 1.2H), 5.07 (s, 0.8H), 4.52(s, 0.8H), 4.27 (s, 1.2H), 4.12˜4.02 (m, 2.4H), 3.94˜3.83 (m, 1.6H).

Example 77−{[2-N-(Benzhydryloxycarbonyl)-guanin-1-yl]acetyl}-1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one

[0272] To a solution of4-[2-N-(bezhydryloxycarbonyl)-guanin-9-yl]-acetic acid (1.26 g, 3.0mmol), 1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCI salt(1.07 g, 3.0 mmol), and PyBOP (1.87 g, 3.6 mmol) in DMF (10 mL) wasadded N,N-diisopropylethylamine (1.05ml, 6.0 mmol) at 10 ° C. Afterstirring for additional stirring for 1 h, the reaction mixture wasconcentrated under reduced pressure and the residue was dissolved inethyl acetate. The solution was washed with water, saturated sodiumbicarbonate solution, 20% aqueous citric acid solution, and brine. Theorganic layer was dried over magnesium sulfate and evaporated in reducedpressure. The residue was purified by column chromatography to affordthe titled compound (1.25 g, 58%). ¹H NMR (DMSO-d₆) δ 11.63 (br. s, 1H),11.25 (br. s, 1H), 8.43˜8.32 (in, 2H), 8.09 (m, 1H), 7.81 (s, 0.6H),7.76 (s, 0.4H), 7.46˜7.28 (m, 10H), 6.86 (s, 1H), 5.14 (s, 1.2H), 5.06(s, 0.8H), 4.52 (s, 0.8H), 4.27 (s, 1.2H), 4.10˜4.00 (m, 2.4H),3.92˜3.82 (m, 1.6H)

Example 784-{[4-N-(Benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-1-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0273] The title compound (2.53 g, 78%) was synthesized by reaction of1-(2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (679 mg, 2.0 mmol)with [4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetic acid (1.90 g, 5.0mmol) as per the procedure of example 77.

Example 794-{[4-N-(Benzhydryloxycarbonyl)-cytosin-1-yl]acetyl}-1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one

[0274] The title compound (0.56 g, 27%) was synthesized by reaction of1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (1.07 g,3.0 mmol) with [4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetic acid(1.14 g, 3.0 mmol), as per the procedure of example 77. ¹H NMR(DMSO-d₆)δ 10.98(s, 11H), 8.40(s, 1H), 8.32(m, 11H), 8.07(m, 11H), 7.89(d, 0.6H),7.85(d, 0.4H), 7.46˜7.28(m, 1H), 6.96(m, 1H), 6.79(s, 1H), 4.81(s,1.2H), 4.73(s, 0.8H), 4.45(s, 0.8H), 4.27(s, 1.2H), 4.07˜3.97(m, 2.4H),3.90˜3.80(m, 1.6H).

EXAMPLE 804-{[4-N-(Benzhydryloxycarbonyl)-cytosin-1-yl]acetyl}-1-(4-fluoro-2-nitro-benzenesulfonyl)-piperazin-2-one

[0275] The title compound (1.19 g, 60%) was synthesized by reaction of1-(4-fluoro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (1.02 g,3.0 mmol) with [4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetic acid(1.14 g, 3.0 mmol), as per the procedure of example 77. ¹H NMR (DMSO-d₆)δ 10.98 (s, 1H), 8.41 (m, 1H), 8.24 (dd, 1H), 7.87 (m, 2H), 7.50˜7.25(mn, 10H), 6.96 (m, 1H), 6.79 (s, 1H), 4.81 (s, 1.2H), 4.73 (s, 0.8H),4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.06˜3.95 (mn, 2.4H), 3.90˜3.80 (m,1.6H).

Example 814-{[6-N-(Benzyloxycarbonyl)-adenin-9-yl]acetyl}-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0276] The title compound (731 mg, 61%) was synthesized by reaction of1-(2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (644 mg, 2.0 mmol)with [6-N-(benzyloxycarbonyl)-adenin-9-yl]-acetic acid (655 mg, 2.0mmol) as per the procedure of example 77. ¹H NMR (DMSO-d₆) δ 10.69 (s,1H), 9.00 (m, 1H), 8.71 (m, 1H), 8.63˜8.58 (mn, 2H), 8.31 (s, 0.6H),8.29 (s, 0.4H), 7.47˜7.33 (m, 5H), 5.39 (s, 1.2H), 5.29 (s, 0.8H), 5.21(s, 2H), 4.57 (s, 0.8H), 4.29 (s, 1.2H), 4.15˜4.09 (m, 2.4H), 3.93˜3.86(m, 1.6H).

Example 824-{[2-N-(Benzyloxycarbonyl)-guanin-9-yl]acetyl}-1-(2-nitro-benzenesulfonyl)-piperazin-2-one

[0277] To a solution of [2-N-(benzyloxycarbonyl)-guanin-9-yl]-aceticacid (1.48 g, 4.5 mmol), 1-(2-nitro-benzenesulfonyl)-piperazin-2-one HClsalt (1.45 g, 4.5 mmol), and PyBOP (2.81 g, 5.4 mmol) in DMF (13 mL) wasadded N,N-diisopropylethylamine (1.96 mL, 13 mmol) at room temperature.After stirring for additional 40 min, the reaction mixture was dilutedwith water (80 mL) to precipitate solid. The solid was filtered off,washed with ethyl alcohol and triturated with acetonitrile and THF togive pure product (1.56 g, 58%). ¹H NMR (DMSO-d₆) δ 11.47 (s, 1H), 11.38(s, 1H), 8.40˜7.96 (m, 4H), 7.82 (s, 0.6H), 7.78 (s, 0.4H), 7.45˜7.35(m, 5H), 5.25 (s, 2H), 5.13 (s, 1.2H), 5.04 (s, 0.8H), 4.51 (s, 0.8H),4.26 (s, 1.2H), 4.10˜4.02 (m, 2.4H), 3.93˜3.83 (m, 1.6H).

Example 834-{[2-N-(Benzyloxycarbonyl)-guanin-9-yl]acetyl}-1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one

[0278] The title compound (1.32 g, 60%) was synthesized by reaction of1-(4-chloro-2-nitro-benzenesulfonyl)-piperazin-2-one HCl salt (1.25 g,3.5 mmol) with [2-N-(benzyloxycarbonyl)-guanin-9-yl]-acetic acid (1.15g, 3.5 mmol) as per the procedure of example 82. ¹H NMR (DMSO-d₆): δ11.46 (s, 1H), 11.37 (s, 1H), 8.44˜8.31 (m, 2H), 8.09 (m, 1H), 7.82 (s,0.6H), 7.78 (s, 0.4H), 7.46˜7.33 (m, 5H), 5.25 (s, 2H), 5.12 (s, 1.2H),5.04 (s, 0.8H), 4.51 (s, 0.8H), 4.26 (s, 1.21H), 4.10˜4.00 (mn, 2.4H),3.92˜3.82 (m, 1.6H).

Example 843-Methyl-1-(2-nitro-benzensulfonyl)-4-[(thymin-1-yl)acetyl]-piperazin-2-one

[0279] To a solution of (thymin-1-yl)-acetic acid (55 mg, 0.30 mmol),1-(2-nitro-benzenesulfonyl)-3-methyl-piperazin-2-one HCl salt (100 mg,0.30 mmol), and PyBOP (156 mg; 0.45 mmol) in DMF (1 mL) was addedN,N-diisopropylethylamine (0.08 mL) at room temperature. After stirringfor additional 2 h at 40° C., the reaction mixture was concentratedunder reduced pressure. The residue was dissolved in dichloromethane andwashed with water. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated in reduced pressureand the residue was purified by column chromatography to give the titledcompound (100 mg, 72%). ¹H NMR (500 MHz; DMSO-d₆) δ 11.31 (s, 1H), 8.33(d, 1H), 8.10 (d, 1H), 8.04˜7.93 (m, 2H), 7.35 (s, 1H), 4.85˜4.60 (m,2.5H), 4.55˜4.40 (m, 0.5H), 4.20˜4.10 (m, 0.5H), 4.05˜3.90 (m, 1.5H),3.78˜3.60 (m, 1H), 3.30 (m, 1H), 1.74 (s, 3H), 1.42 (d, 1H), 1.27 (d,2H).

Example 851-(2-Nitro-benzensulfonyl)-3-phenyl-4-[(thymin-1-yl)acetyl]-piperazin-2-one

[0280] To a solution of (thymin-1-yl)-acetic acid (13 mg, 0.071 mmol),1-(2-nitro-benzenesulfonyl)-3-phenyl-piperazin-2-one HCl salt (28 mg,0.070 mmol), and PyBOP (40 mg, 0.077 mmol) in DMF (0.3 mL) was addedN,N-diisopropylethylamine (0.018 mL) at room temperature. After stirringfor additional 27 h, the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography to give thetitled compound (16 mg, 43%). ¹H NMR(500 MHz ; DMSO-d₆) δ 11.33 (s, 1H),8.36 (d, 1H), 8.13 (d, 1H), 8.05˜7.94 (m, 3H), 7.39˜7.29 (m, 5H), 5.93(s, 1H), 4.87 (d, 1H), 4.67 (d, 1H), 4.22˜4.16 (m, 2H), 3.96 (m, 1H),3.78 (m, 1H), 1.74 (s, 3H).

[0281] All of the references cited herein are incorporated by referencein their entirety.

[0282] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention specifically described herein.Such equivalents are intended to be encompassed in the scope of theclaims.

What is claimed is:
 1. A compound having formula I:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.
 2. The compound of claim 1, wherein R6 is H or protected or unprotected side chain of natural α-amino acid.
 3. The compound of claim 1, wherein B is thymine (T), cytosine (C), adenine (A), or guanine (G).
 4. The compound of claim 1, wherein the protecting group of B is benzyloxycarbonyl or benzhydryloxycarbonyl.
 5. The compound of claim 1, wherein R1 is nitro and R3 is halogen, trifluoromethyl, or methyl; and R2, R4, R5 are H.
 6. The compound of claim 1, wherein R1 is nitro, R3 is Cl, R5 is Cl or methyl, and R2 and R4 are H.
 7. The compound of claim 1, wherein R3 is nitro, and R1, R2, R4 and R5 are H.
 8. The compound of claim 1, wherein R3 is nitro, R1 is Cl, and R2, R4 and R5 are H.
 9. A method of making the compound of claim 1, comprising cyclizing a compound of formula VI in the presence of a coupling reagent that is customarily used in peptide synthesis or mixed anhydride, wherein the formula VI is represented as follows:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.
 10. A method of making the compound of claim 1, comprising coupling reaction of a compound of formula IV with a nucleobase acetic acid moiety in the presence of non-nucleophilic organic base and a coupling reagent that is customarily used in peptide synthesis, wherein said formula IV is represented as follows:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid, wherein said nucleobase acetic moiety is represented as follows:

wherein B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.
 11. A compound having formula V

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; R7 is H, (C₁-C₄) alkyl, or aryl; and B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.
 12. The compound of claim 11 wherein R7 is methyl, ethyl, or t-butyl.
 13. A method of making the compound of claim 11, comprising coupling reaction of a compound of formula II with a nucleobase acetic acid moiety in the presence of non-nucleophilic organic base and a coupling reagent that is customarily used in peptide synthesis, wherein formula II is represented as follows:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄alkyl, nitro, nitrile, C₁-C₄alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; R7 is H, (C₁-C₄) alkyl, or aryl, wherein said nucleobase acetic moiety is represented as follows:

wherein B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.
 14. A compound having formula II

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄alkyl, nitro, nitrile, C₁-C₄alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and R7 is H, (C₁-C₄) alkyl, or aryl.
 15. A method of making the compound of claim 14, comprising coupling reaction of N-(2-aminoethyl)-glycine derivative with sulfonyl chloride derivative in the presence of non-nucleophilic organic base, wherein the N-(2-aminoethyl)-glycine derivative is represented as follows:

wherein R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and R7 is H, (C₁-C₄) alkyl, or aryl, wherein the sulfonyl chloride derivative is represented as follows:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro.
 16. A method of making the compound of claim 14, comprising reacting 2-aminoethyl sulfanylamide derivative with haloacetate derivative by a nucleophilic substitution reaction in the presence of non-nucleophilic organic base, wherein 2-aminoethyl sulfanylamide derivative is represented as follows:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄alkyl, nitro, nitrile, C₁-C₄alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro, and wherein the haloacetate derivative is represented as follows:

wherein R7 is H, (C₁-C₄) alkyl or aryl; and X is Cl, Br, or I.
 17. A compound having formula IV and its free acid form

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and HY is organic or inorganic acid.
 18. The compound of claim 17, wherein HY is HCl or TFA.
 19. A method of making the compound of claim 17, comprising cyclizing a compound of formula III in the presence of a coupling reagent that is customarily used in peptide synthesis or mixed anhydride, followed by deprotection of t-Boc in acid, wherein said formula III is represented as follows:

wherein R1, R2, R3, R4, R5 is independently H, halogen, C₁-C₄ alkyl, nitro, nitrile, C₁-C₄ alkoxy, halogenated C₁-C₄ alkyl, or halogenated C₁-C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro; and R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid.
 20. A method of making PNA oligomer, comprising linking together the compound of claim
 1. 